Immunotherapeutics for biodefense

ABSTRACT

Human neutralizing antibodies (full-length or functional fragments) are useful as anti-toxins or anti-infectives with respect to infective agents such as, for example, anthrax, botulinum, smallpox, Venezuelan equine encephalomyelitis virus (VEEV), West Nile virus (WNV) and the like.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 10/364,743 filed Feb. 11, 2003 which, in turn, claims priority to U.S. Provisional Application Nos. 60/356,086, 60/376,408 and 60/428,807 filed on Feb. 11, 2002, Apr. 29, 2002 and Nov. 25, 2002, respectively.

BACKGROUND

[0002] 1. Technical Field

[0003] This disclosure relates to human neutralizing antibodies (full-length or functional fragments) useful as anti-toxins or anti-infectives with respect to infective agents such as, for example, anthrax, botulinum, smallpox, Venezuelan equine encephalomyelitis virus (VEEV), West Nile virus (WNV) and the like.

[0004] 2. Background of Related Art

[0005] Concerns over our capacity to prevent and treat anthrax infection have been high following the recent acts of terrorism in the US. Though a vaccine for anthrax exists, it consists of six spaced inoculations, requires a yearly booster, and produces unpleasant side effects in most vaccinees. This has kept it from widespread use, and is still a major drawback to inoculating the general public. The anthrax vaccine presently in use is made by Bioport (Lansing, Mich.) through a process that involves purifying the protective antigen out of a lysate of Bacillus anthracis. It appears that this is still the Sterne live vaccine strain, though there are other strains lacking the LF and EF that might be used to generate this vaccine, as well as high yielding recombinant Bacillus subtilis strains that could be used. Presumably the difficulties of testing and comparing such additional vaccines, and the small market, have hindered actual testing, licensing and production. The side effects of the present vaccine, its connection in the minds of many with Gulf War Syndrome, and the possibility that large scale vaccination may now be desirable in the face of terrorist threats suggests that improvements in the present vaccine will now be pursued. As long as there is no reasonable vaccine, treatment for exposure remains the primary response to acts of terrorism.

[0006] The recent anthrax exposures in the United States from contaminated letters have all involved strains of anthrax susceptible to antibiotics, but tragically, a number of people died due to delayed diagnosis. In light of this, it would be useful to have a treatment for anthrax exposure that could prevent illness and death and allow antibiotic therapy and/or adaptive immunity additional time to be effective.

[0007] The primary cause of death from anthrax is the reaction of the body to two related toxins produced by the bacteria. These both contain a processed protein called PA63 that binds as PA83 to cellular receptors, whereupon it is processed to PA63. The receptor on human cells for anthrax toxin, the ATR (anthrax toxin receptor) was recently identified. See Bradley et al., Nature, Vol 414, Nov. 8, 2001. PA63 then forms a heptamer that is capable of binding with either the EF protein (edema factor) or the LF protein (lethal factor). Endosomal internalization of heptamerized PA63 and bound EF and/or LF facilitates the introduction of the EF and LF toxins into the cell. Acidification of the endosomal vesicle causes the PA heptamer to form a pore through which the EF and/or LF can enter the cytosol, where they exert their toxic effects. None of the three components, EF, LF, or PA, can cause illness by itself.

[0008] Several lines of evidence indicate that it is possible to prevent receptor binding of PA or to obstruct the interaction of EF and LF with PA. The vaccine itself uses the purified PA moiety alone to create antibodies that are protective. Little et al. (Infect. Immun. 65:5171-5175 (1997)) passively administered PA antibodies to guinea pigs that then showed protection against subsequent anthrax infection—70% protection for polyclonal antibodies, and a two day delay of death for one monoclonal. Single-chain antibody fragments (scFvs) have also been used to inhibit receptor binding by PA. Cirino et al. (Infect. Immun. 67:2957-2963 (1999)) identified a number of scFvs from a naive human library that bound PA83. They then used these in a cell-based assay in which PA32, a truncated version of PA63, was fused with EGFP, and was taken up by cells in a similar manner to PA63. The fluorescence of EGFP could then be used to monitor the effect of these scFvs against PA32-EGFP in cellular uptake. One scFv was identified which could prevent the uptake of the PA32-EGFP by the cell. Mourez et al. (Nature Biotech. 19:958-961 (2001)) created a polyvalent peptide inhibitor against the anthrax toxin that binds to the PA63 heptamer at or near the EF/LF site. They used cell-based assays to demonstrate that this inhibitor can protect cells against PA/LF toxicity. They also showed that rats could be challenged with 10 times the minimum lethal dose of PA and LF and still be protected when the inhibitor was introduced 3-4 minutes after challenge.

[0009] These data all suggest that it should be possible to develop a therapeutic human combinatorial antibody for combination therapy with antibiotics in patients with late diagnoses of anthrax infection.

[0010] Venezuelan equine encephalomyelitis virus (VEEV) is a mosquito-borne alphavirus which can be transmitted to both equine and human hosts. Whereas infection of horses and donkey populations can result in large mortalities, natural human infection usually consists of fever, chills, malaise, and severe headache with only 1-4% of people progressing to severe encephalitis. However, VEEV has been classified as a “Category B” critical biological agent by the CDC due to its low human infective dose, easy production, and ability to be aerosolized. Potentially, aerosolized VEEV could be used as an effective bioweapon using forms of VEEV that are known to be highly infectious and that can easily gain direct access to the central nervous system via the olfactory tract. Once replication of the virus occurs in the CNS, encephalitis is a serious risk. Unfortunately, treatment of VEEV infection is limited to supportive care.

[0011] There are investigational vaccines available against VEEV, although their use is limited to laboratory workers at risk and military troops. A live attenuated vaccine, TC-83 (FDA Investigational New Drug #142) (Pittman et al., Vaccine 14, pp337-343 (1996)), has been used in both these settings. This vaccine was established by serial passage of the virulent Trinidad donkey virus in tissue culture. TC-83 virus elicits VEEV-specific neutralizing antibodies in most humans and equines. (Kinney et al., Virology 170, 19-30 (1989)). In laboratory animals, the vaccine was able to produce immunity to subcutaneous or airborne challenge with virulent VEEV strains (Phillpotts, Vaccine 17, pp2429-2435(1999)). However, up to 18% of human vaccinees fail to develop protection from the initial vaccinations. In addition, the vaccine has a relatively high rate of reactogenicity (25%). One recent report states that TC-83 is no longer available for human use (Philipofts et al., Vaccine 20, p1497-1504 (2002)). Concerns over TC-83 prompted the development of an inactivated vaccine, C-84. However, C-84 did not produce protection against aerosol challenge with virulent strains of the virus in animal models (Pittman et al., Vaccine, 14, pp337-343 (1996).). As a result, C-84 is not used as a primary immunogen for laboratory workers, rather its usefulness is as a follow up vaccine for non-responders to TC-83 or as a booster where it serves as a recall antigen. There is, therefore, an urgent need for anti-VEEV therapies, such as potent neutralizing anti-VEEV antibodies.

[0012] VEEV is an enveloped virus, where the envelope and capsid structures are separated by a lipid bilayer and are thought to interact through the membrane-spanning tail of the E2 glycoprotein. Similar to Sindbis virus, VEEV has virion protein spikes organized as trimers of E1/E2 heterodimers (Paredes, et al., J. Virology, 75, ppp9532-9537 (2001); Phinney, et al. J Virology, 74, pp5667-5678 (2000)). The epitopes present on E1 (gp50) and E2 (gp56) that may be involved in the critical neutralization sites have been studied using monoclonal Abs (Mathews and Roehrig, J. Immunology, pp2763-2767 (1982)). Site E2^(c) is present at the tip of the E2 spike and believed to be the neutralizing (N) epitope. Additional epitopes have also demonstrated neutralizing activity and may have a close structural relationship with the E2^(c) site.

[0013] The mouse model of VEEV infection is believed to follow a pathogenesis of disease that is similar to that in humans. Passive transfer of neutralizing Ab prior to viral challenge has effectively prevented death in these normal mice (see for example, Roehrig and Mathews, Virology (1985) 142, pp 347-356; Phillpotts, et al., Vaccine (2002) 20, 1497-1504). Non-neutralizing Abs have also shown protection in i.p. or i.v. viral challenge of mice (Hunt and Roehrig, Vaccine (1995) 13, pp281-288; and Hunt et al., Virology (1991) 185, 281-290). Although the mechanism of non-neutralizing Ab protection from viral challenge is not known, it is surmised that they may act by delaying viral replication and in doing so allow the host immune system time to respond to and control the viral infection (Hunt et al., Virology (1991) 185, 281-290). An effective therapy for humans against airborne exposure to VEEV may require a faster mode of action, such as direct neutralization of the virus at, or close to, the time of exposure. Of particular concern is the ability of the neutralizing Ab to prevent the spread of VEEV to the brain. In this regard, it is significant that administration of a neutralizing Ab to mice up to 24 hours after airborne viral challenge showed protective effects (Phillpotts, et al., Vaccine, 20, 1497-1504 (2002)).

[0014] The murine antibodies described in these and similar studies might be of use in the prevention and treatment of VEEV infection in humans. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical applications, especially when repeated administration is required for therapy. A process termed antibody humanization can be used to decrease the immunogenicity of a rodent antibody by replacing most of the rodent antibody with human antibody regions while striving to maintain the original antigenic specificity. However, this undertaking is usually time-consuming and costly and does not rule out the possibility of an immunogenic response to the humanized Ab. Antibodies that are fully human and target neutralizing epitopes on VEEV are the most desirable therapeutic candidates, as they pose the best chance of an effective block of viral infection and present the least risk of being immunogenic.

[0015] Botulinum neurotoxin is one of the most potent bacterial toxins known, with an LD50 for humans of 1 ng/kg. The toxin is produced by the bacteria Clostridium botulinum, as well as by several other Clostridium species, and seven serotypes of toxin (A through G) have been recognized. On a molecular level, the toxin is produced as a 150 kDa protein that is cleaved by exposure to proteases to generate two chains that remain associated: a light chain, of about 50 kDa, and a heavy chain, of 100 kDa. The heavy chain contains the domain responsible for binding to neuronal cells, while the light chain contains a zinc-dependent endoprotease domain that enters the neuronal cytosol. Once inside, this endoprotease exerts its toxic effect by proteolytic cleavage of synaptic proteins, including synaptobrevins, syntaxin and SNAP-25. Destruction of these proteins inhibits neurotransmission and results in a progressive paralysis and death.

[0016] Antibodies against botulinum neurotoxin have been shown to be protective in passive and active immunization models. The PBT vaccine, consisting of serotypes A through E, is currently made available by the Department of Defense and the Centers for Disease Control to people at risk for exposure to botulinum neurotoxin. Serotypes D and G are rarely encountered in natural human infections, though serotype F is common, and is lacking in the PBT vaccine. The possibility of serotypes D and G being utilized in a bioterrorist attack should not, however, be overlooked. For natural infections, polyclonal antibody preparations have been successfully used as immunotherapeutics, but they must be given early in infection to minimize the entry of toxin into the neuronal cells. Several polyclonal immunoglobulin preparations are available as immunotherapeutics: an equine trivalent (A, B, and E) preparation, an equine heptavalent preparation with the Fc portion of the immunoglobulins removed by proteinase cleavage, and a human immunoglobulin preparation (hBIG) obtained from donors vaccinated with the PBT vaccine. Both equine preparations have had difficulties with hypersensitivity reactions in treated individuals. The human preparation is well-tolerated and effective, but it is in short supply and only useful against five of the seven serotypes. Even for natural infections, it would be useful to have a ready supply of fully human neutralizing antibodies to all the serotypes of botulinum neurotoxin. The heightened awareness of our vulnerability to biological terrorism following the intentional anthrax release of 2001 makes it even more critical to develop such immunotherapeutics.

[0017] Since the declaration of smallpox eradication in May 1980 (Fenner et al., 1998) and the cessation of vaccination programs, immunity has waned among those vaccinated, and those born since 1980 are unvaccinated. The world-wide lack of immunity dramatically increased the threat of a deliberate release of variola virus, the causative agent of smallpox, as a bioweapon. The variola virus has characteristics that make it particularly suitable for biological warfare. The virus can spread from person to person by the respiratory route or by direct contact. An aerosol release of the virus can disseminate widely, because of its considerable stability in aerosol form and because the infectious dose can be very small (Wherle et al., 1970). There is no specific treatment for the disease. There is also a threat that a large quantity of infectious virus may be missing. Alibek (Alibek, 1999), a former deputy director of the Soviet Union's civilian bioweapons program, reported that beginning in 1980, the Soviet government initiated a bioweapons program and developed a method to produce many tons of variola virus annually for transport in bombs and ballistic missiles. With the decline of financial support for and the discontinuation of the Soviet civilian biowarfare program in 1992, experienced scientists, equipment, and materials may have been transferred into other countries. The reported epidemics in Asia had a mortality-rate of 30% or more (Fenner et al., 1998). Today, with a more susceptible and highly mobile population, the virus can spread very rapidly and widely throughout the country and the world.

[0018] Variola virus is a DNA virus, a member of the family Poxviridae and the genus orthopoxvirus (Fenner et al., 1998) that includes vaccinia, monkeypox virus, and several other animal poxviruses that cross-react serologically. Only variola virus can readily transmit from person to person (reviewed in Breman and Henderson, 2002). DNA sequence analysis revealed that variola and vaccinia viruses are closely related (Massung et al., 1994). The infectious dose of variola virus is believed to be very low, only a few virions (Wherle et al., 1970). It transcribes and replicates its genome and assembles progeny virions entirely within the cytoplasm of infected cells (reviewed in Moss, 1996). Four types of infectious forms are produced: intracellular mature virus (IMV), the intracellular enveloped virus (IEV), the cell-associated enveloped virus (CEV), and the extracellular enveloped virus (EEV) (reviewed in Moss 1996). IMV is the major form that remains in the cytoplasm. EEV represents a minor fraction of infectious particles but is the biologically relevant form in terms of long-range dissemination and spread of the virus in vitro and in vivo (Payne, 1980; Smith and Vanderplasschen, 1998; Law and Smith, 2001). It has been shown that an immune response against EEV but not IMV is necessary for protection against orthopoxvirus infection (Appleyard et al., 1971; Boulter, 1969; Boulter and Appleyard, 1973; Boulter et al., 1971; Ichihashi et al., 1971; Morgan, 1976; Payne, 1980; Payne and Kristensson, 1985; Turner and Squires, 1971). Six genes are reported to encode ten proteins for EEV outer envelope (Payne, 1978; Payne, 1979). They are A33R (gp22-28) (Roper et al., 1996), A34R (gp22-24) (Duncan and Smith, 1992), A36R (p45-50) (Parkinson and Smith, 1994), A56R (gp86, a heavily glycosylated hemagglutinin) (Payne and Norrby, 1976; Shida, 1986), B5R (gp42) (Isaacs et al., 1992; Englestad et al., 1992), and F12L or F13L (p37) (Hirt et al., 1986; Blasco and Moss, 1991). Recently A36R protein was found to be absent in the CEV and EEV particles (van Eijl et al., 2000). Envelope proteins of IMV are A27L (p14) (Rodriguez and Esteban, 1985), D8L (p32) (Maa et al., 1990; Niles and Seto, 1988), A17L (p21) (Rodriguez et al., 1995), and L1R (M25, a myristylated virion protein) (Franke et al., 1990). A27L, A17L and L1R are implicated in the fusion and penetration of IMV (Ichihashi and Oie, 1996).

[0019] The smallpox vaccine, manufactured from the vaccinia virus, was the first vaccine ever produced. The current stockpile consists of a live vaccinia virus that was grown on the skin of calves. In the United States, the reserve supply is limited; there is just enough to vaccinate 6 to 7 million people. None of the other countries have enough doses to cover their population if an outbreak occurs. Smallpox vaccination is also associated with more severe adverse effects than any other type of vaccination, which was one of the reasons for ending vaccination after eradication (Ober et al., 2002). Presently, it is recommended for use only in suspected cases and not for mass vaccination by World Health Organization and United States, Centers for Disease Control and Prevention (Smallwood et al., 2002). Vaccination with vaccinia virus is effective in preventing smallpox for at least five years and may prevent or modify infection for a much longer period, but this varies greatly from person to person.

[0020] There is general agreement that neutralizing antibodies play an important role in immunity against orthopox viruses, particularly in the prevention of reinfection and dissemination of infection. The benefit of vaccinia immune globulin (VIG) in preventing infection or controlling adverse effects from vaccinia immunization have been clearly documented (Kempe, 1960, Kempe et al., 1961, Hobday, 1962). Polyclonal antiserum against the recombinant B5R protein inhibited EEV infection (Galmiche et al., 1999). Mice vaccinated with B5R protein were protected against a lethal challenge with vaccinia virus that is likely to be mediated by neutralizing antibodies. Protein A33R but not A34R and A36R was also protective in active and passive immunization but protection did not correlate with antibody titers and anti-A33R antibodies did not neutralize EEV in vitro. The authors stated the protection probably involves a mechanism different from simple antibody binding (Galmiche at al., 1999, Schmaljohn et al., 1999). Prophylactic as well as therapeutic administration of mouse neutralizing antibody against the trimeric 14 kDa protein (A27L, p14) of vaccinia virus localized in the membrane of the IMV effectively controlled the replication of the virus in mice (Ramirez et al., 2002). DNA vaccination with L1R and A33R genes protected mice against a lethal virus challenge with neutralizing antibodies to L1R and A33R (Hooper et al., 2000).

[0021] As described in the recent issue of Emerging Infectious Diseases (Casadeval, 2002), the only available countermeasure that can provide immediate immunity against a biological agent is passive immunization with antibodies. Vaccine takes time to induce protective immunity and depends on the host's ability to mount an immune response, whereas passive immunization can theoretically confer protection regardless of the immune status of the host. Low cytotoxicity and highly specific activity are among the advantages of passive immunization over other measures of postexposure treatment.

[0022] Identification of immune donors with good serum neutralizing activity and the construction of combinatorial antibody libraries from the bone marrow of such donors is a logical approach for the isolation of a large panel of highly specific neutralizing antibodies to viral infection (Burton et al.,1991; Barbas et al., 1992; Williamson et al., 1993; Burioni et al., 1994; Maruyama et al., 1999; Maruyama et al., 2002). The selection of libraries on recombinant envelope proteins containing neutralizing epitopes is straightforward. Unlike mouse antibodies, human antibodies are non-immunogenic and once their efficacy is fully characterized in susceptible animals, they can be safely administered to patients.

[0023] It would be desirable to identify antibodies that neutralize infective agents of the types that may be employed in bio-warfare. It would also be advantageous if these bio-defense antibodies could be derived from a single antibody library.

SUMMARY

[0024] Using phage display technologies and messenger RNA derived from lymphoid cells of vaccinated or convalescent humans, it is possible in accordance with the methods described herein to rapidly identify panels of antibody fragments (Fabs) that bind to antigens from infective agents. The strength of the interaction of these Fabs with antigen can be determined by studying their binding kinetics using surface plasmon resonance. These human Fabs can be readily converted to full-length IgG by subcloning into appropriate mammalian expression vectors containing the remaining constant region domains. Testing of Fabs or antibodies from these panels in viral or toxin inhibition studies in vitro and in vivo in small animal models can then identify a subset of neutralizing antibodies that will be suitable for continuation to pre-clinical and clinical testing. These antibodies may then be used as immunotherapeutics in the treatment of individuals infected with or exposed to any of the above agents, or may be used prophylactically in individuals expected to be at risk for exposure.

[0025] Thus, in one aspect, an antibody library is described from which antibodies or functional fragments thereof can be identified, isolated and produced in large quantities to neutralize or prevent infection by an infective agent.

[0026] In another aspect, heterodimeric antibodies are described which are effective in treating anthrax infection. The heterodimeric antibodies are selected from an antibody library. The library is preferably generated from an immunized human source. The heterodimeric antibodies bind to and disable the activity of a molecule involved in anthrax infection, such as, for example, the anthrax protective antigen or the EF or LF proteins and thereby inhibit toxin activity by interfering with the processes involved in toxin introduction to the cell. These processes include but are not limited to the following: PA83 binding to receptor, PA83 processing to PA63, PA63 interaction to form a prepore complex, EF or LF binding to the prepore, prepore conformational changes permitting membrane translocation of EF or LF, or EF or LF translocation through the pore. This interference is such that the toxic effects associated with uptake of these proteins by cells in the body are slowed or eliminated. In particularly useful embodiments, the heterodimeric antibodies have an affinity of at least 1×10⁻⁸ M for a molecule involved in anthrax infection. In another embodiment, these antibodies can be used as diagnostic reagents.

[0027] In another aspect, antibodies or functional fragments of antibodies that neutralize Botulinum are described.

[0028] In another aspect, antibodies or functional fragments of antibodies that neutralize Variola virus (Small Pox)/Vaccinia virus are described.

[0029] In another aspect, antibodies or functional fragments of antibodies that neutralize Venezuelan Equine Encephalomyelitis Virus (VEEV) are described.

[0030] In another aspect, antibodies or functional fragments of antibodies that neutralize West Nile virus (WNV) are described.

[0031] In another aspect, antibodies or functional fragments of antibodies that neutralize Dengue are described.

[0032] In another aspect, methods of prophylactically administering antibodies or functional fragments of antibodies are described to prevent infection by an infective agent.

[0033] In another aspect, methods of administering antibodies or functional fragments of antibodies are described to treat infection by an infective agent.

[0034] In another aspect, antibodies that have Fab components that neutralize infective agents sub-stoichiometrically are described.

[0035] In yet another aspect, a vaccine that contains a multimer of PA63 is described, as well as methods of using such a vaccine.

[0036] In another aspect, an antibody or antibody fragment having binding affinity for an infective agent in accordance with this disclosure are used in an assay to detect the presence of an infective agent (either directly or by detecting a toxin released by the infective agent) to diagnose the presence of a disease associated with the infective agent.

[0037] In another aspect, an antibody or antibody fragment having binding affinity for an antibody to an infective agent in accordance with this disclosure is used as a control antibody in an assay to detect the presence of antibodies in response to exposure to an infective agent. Such assays are useful in detecting exposure to an infective agent and diagnosing a disease associated with the infective agent.

[0038] In another aspect, kits for diagnosing a disease associated with an infective agent are described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 is a table summarizing the exposure history of individuals suitable as a source of tissue for library generation in accordance with preferred embodiments of the present disclosure.

[0040]FIG. 2 shows titers of bone marrow and blood donors to PA83 antigen of Anthrax.

[0041]FIG. 3 shows the sequence analysis of the VH positive reactivity to PA63 and PA83.

[0042]FIG. 4 shows the sequence analysis of the VK positive reactivity to PA63 and PA83.

[0043]FIG. 5 shows the sequence analysis of the VL positive reactivity to PA63 and PA83.

[0044]FIG. 6 shows sequences of variant human kappa light chains of antibodies to the anthrax proteins PA83 and PA63.

[0045]FIG. 7 shows sequences of variant human lambda light chains of antibodies to the anthrax proteins PA83 and PA63.

[0046]FIG. 8A-8C show amino acid sequences of variant human heavy chains of antibodies to the anthrax proteins PA83 and PA63.

[0047]FIG. 9 shows neutralization of Anthrax toxin activity by purified Fabs.

[0048]FIG. 10 shows the percent protection (compared to toxin alone) for seven serially diluted Fabs.

[0049]FIG. 11 shows Western blots demonstrating the ability of Fabs produced in accordance with the methods described herein to react with linear epitopes on PA63 and/or PA83. All of the five anti-PA83 Fabs tested appear to bind to linear epitopes on PA83 while the anti-PA63 antibody, in contrast does not bind to denatured PA63, and shows what appears to be faint, presumably non-specific binding to PA83.

[0050]FIG. 12 shows an ELISA titration of selected Fabs on PA83 and PA63. Cleavage to PA63 dramatically alters the binding of FML8E and F9L6R2, but FMK7C binds equally well to both forms. F951L631D binds only to PA63. Maximum binding seen is ¼ that of FMK7C, suggesting that only a portion of the PA63 material is in a form with which F951L631D can interact.

[0051]FIG. 13 shows the result of testing wherein a his tagged version of Fab FML8E was used in competition with other untagged Fabs to assess epitope specificity.

[0052]FIG. 14 shows an ELISA titration of selected Fabs at 1 μg/ml against PA63 and PA83 at 200 ng/well.

[0053]FIG. 15 shows the competition of two Fabs, 63L1D and 83K7C, with LF for binding against PA63.

[0054]FIG. 16 shows the competition of two anti-PA83 Fabs, 83K7C and 83L8E, with mouse monoclonal antibody 14B7.

[0055]FIG. 17 shows the results of an assay to determine whether selected Fabs could neutralize the effect of toxin after PA had bound to cells.

[0056]FIG. 18 shows the results of testing Fabs 83K7C and 63L1D in vivo against recombinant toxin challenge.

[0057]FIG. 19 shows serum reactivity on immobilized TC-83 antigen of VEEV.

[0058]FIGS. 20A through 20D show the results of screening of Fab clones from four libraries (951K. 951L, 1037K and 1037L) for binding to immobilized TC-83 of VEEV.

[0059]FIG. 21 shows direct titration of purified human Fabs on immobilized TC-83 antigen of VEEV.

[0060]FIG. 22 shows competition of the human Fabs against the murine Fab mHy4 (3B4C-4) for binding to immobilized TC-83 antigen of VEEV or BSA.

[0061]FIGS. 23A and 23B show the sequences for fully-human Fabs produced in accordance with this disclosure that neutralize VEEV.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0062] The human antibodies in accordance with this disclosure can be whole antibodies or antibody fragments. The antibodies can be heterodimeric or single chain antibodies. The term “heterodimeric” means that the light and heavy chains of the antibody or antibody fragment are bound to each other via disulfide bonds as in naturally occurring antibodies. Single chain antibodies have the light and heavy chain variable regions of the antibody connected through a linker sequence.

[0063] The present human antibodies are identified by screening an antibody library. Techniques for producing and screening an antibody library are within the purview of one skilled in the art. See, Rader and Barbas, Phage Display, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000), U.S. Pat. No. 6,291,161 to Lerner et al. and copending WO 03/025202 and U.S. Provisional Application No. 60/323,400, the disclosures of which are incorporated herein in its entirety by this reference.

[0064] Generally, the first step in producing an antibody library in accordance with this disclosure involves collecting cells from an individual that is producing antibodies against one or more infective agents or antigens from infective agents. Typically, such an individual will have been exposed to the infective agent and/or antigen from an infective agent. In particularly useful embodiments, the individual has been exposed to a plurality of infective agents or antigens from infective agents that are strategically important with respect to biowarfare. Such materials include agents selected from the group consisting of anthrax, antigens from anthrax, botulinum, antigens from botulinum, smallpox, antigens from smallpox, Venezuelan equine encephalomyelitis virus (VEEV), antigens from VEEV, dengue, antigens from dengue, typhoid, antigens from typhoid, yellow fever, antigens from yellow fever, hepatitis, antigens from hepatitis, West Nile virus (WNV), antigens from WNV and the virus responsible for severe acute respiratory syndrome (SARS). FIG. 1 is a table summarizing the exposure history of individuals suitable for use in preparing antibody libraries in accordance with preferred embodiments of the present disclosure. Cells from tissue that produces or contains antibodies are collected from the individual about 7 days after infection or immunization. Suitable tissues include blood and bone marrow.

[0065] Once the cells are collected, RNA is isolated therefrom using techniques known to those skilled in the art and a combinatorial antibody library is prepared. In general, techniques for preparing a combinatorial antibody library involve amplifying target sequences encoding antibodies or portions thereof, such as, for example the light and/or heavy chains using the isolated RNA of an antibody. Thus, for example, starting with a sample of antibody mRNA that is naturally diverse, first strand cDNA can be produced to provide a template. Conventional PCR or other amplification techniques can then be employed to generate the library.

[0066] Screening of the antibody library can be achieved using any known technique such as, for example, by panning against a desired viral antigen. See Rader and Barbas, Phage Display, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000). Certain antigens have been cloned and can be produced recombinantly for use as immunogens. Neutralizing ability can be assessed in cellular assays that determine the ability of the antibody to block the binding of the virus with cellular receptors. Once antibodies having in vitro neutralizing ability are identified, they can be tested in vivo in animal models.

[0067] Antibodies identified in this manner advantageously provide an effective treatment for infection by an infective agent. Because the present antibodies are fully human antibodies, they are safe and easily tolerated. In addition, multiple doses can be given without rapidly raising an anti-idiotype response. Where full length antibodies are used, the higher avidity and larger size (compared to single chain antibodies) may be preferred because they provide greater residence time within the patient's system.

[0068] A particulary useful method for producing antibody libraries in accordance with this disclosure and identifying and characterizing antibodies in accordance with the present disclosure is as follows:

[0069] Libraries.

[0070] Three Fab libraries containing either lambda or kappa light chains and an IgG heavy chain fragment (Fd) were derived from each of two bone marrow samples (951 and 1037, and 1 blood sample (MD3) see FIG. 1) of active military donors immunized against a variety of infectious agents.

[0071] Libraries can undergo selection and screening against a variety of infective agents, such as anthrax, Venezuelan equine encephalitis and botulinum, West Nile virus, vaccinia virus, and dengue.

[0072] Library Creation.

[0073] Total RNA is obtained from bone marrow and blood samples using Tri-reagent BD (Molecular Research Center, Inc.) according to the manufacturer's instructions. Messenger RNA is obtained using Oligotex (Qiagen) spin columns per manufacturer's instructions. Phage libraries expressing antibody Fab fragments (kappa or lambda light chains complexed to the IgG heavy chain fragment (Fd) are constructed in plasmid vectors using the methods described in U.S. application Ser. No. 10/251,085 (the disclosure of which is incorporated herein in its entirety by this reference). Two Fab libraries are generated for each donor, one expressing kappa light chains and one expressing lambda light chains, and all utilizing gamma heavy chains.

[0074] Library Selection.

[0075] Phage bearing Fabs from all of the libraries used are panned through one to four rounds of enrichment against selected viral antigens and toxins. Panning is performed by first incubating a sufficient amount of recombinant antigen (usually 1-2 μg) in 50 μl of Solution A in several Immulon 2 HB microtiter wells overnight at 4° C. Solution A is phosphate buffered saline (PBS), pH 7.4, containing 0.08% boiled casein solution (BC). BC is PBS containing 0.5% casein, 0.01% thimerosal, and 0.005% phenol red. After removal of the antigen solution, wells are blocked for 1 hour at 37° C. with 250 μl of BC containing 1% Tween 20. Phage stocks are diluted into Solution D, consisting of BC with 0.025% Tween 20, and 50 μl are added to each well and incubated for 2 hours at 37° C. Wells are washed ten times with PBS containing 0.05% Tween 20, and then washed once for 2 minutes each with a progressively more acidic series of buffers (D'Mello et al., J Immunological Meth 247:191-203 (2001)): Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl) at pH 5.0, 4.0, and 3.0. Final elution is with 0.1 M glycine-HCl buffer, pH 2.2, 1 mg/ml bovine serum albumen (BSA). The eluent is neutralized with 2M Tris base and added to log phase ER2738 cells. Phage is produced by addition of helper phage (strain VCSM13) to infected bacteria. Individual colonies are generated by infecting susceptible bacteria with phage stock and plating.

[0076] Screening is done with supernatants containing Fab as a fusion protein with a portion of the phage gene III. After screening, positive candidates are sequenced and then subcloned to remove gene III prior to production of Fab for testing. Alternatively, DNA from each panned library can be subcloned to remove the gene III fusion region, and a combination epitope tag can be introduced, consisting of an influenza hemagglutinin epitope tag (HA) (Chen et al., Proc Natl Acad Sci USA 90:6508-12 (1993)) and six histidine amino acids (His tag) for use in subsequent detection and purification by anti-HA and Ni-NTA.

[0077] Library Screening.

[0078] For screening, Fab constructs reactive to the antigen of choice are identified by their ability to bind in an ELISA assay. 100 to 250 ng/well of recombinant antigen in Solution A is incubated overnight in Immulon microtiter dishes and blocked as described above. Screening can be performed in high-throughput by picking 1150 colonies using a Q-pix instrument, and performing ELISAs using a Tecan robot. Individual colonies are grown overnight in deep-well microtiter dishes in a Hi-Gro high-speed incubator shaker. Aliquots are removed and stored with 15% glycerol or 10% DMSO as stocks. After centrifugation of the deep-well dishes, supernatants containing Fab from these stocks are incubated in the wells coated with specific antigens and separately in wells coated with a control antigen such as casein or ovalbumin. Alkaline phosphatase labeled goat anti-human F(ab′)₂ antibody (Pierce) is used to detect Fab bound to antigen. Miniprep DNA (Qiagen) from positive candidates is sequenced by automated dye terminator sequencing (Retrogen, San Diego) in 96 well format across the light and heavy chains using stock primers for these vectors. Sequences are analyzed using DNAstar software to identify and classify unique candidates. From these data a panel of unique variant binders to each recombinant antigen used is determined, and classified into groups of closely related sequences.

[0079] Production and Purification of Fabs from Panels

[0080] Fab Purification.

[0081] For soluble Fab expression and purification, the gene Ill region is removed from unique positive candidates by subcloning. At this point it is also possible to insert an oligonucleotide that will encode a combination epitope tag consisting of an influenza virus hemagglutinin (HA) tag (Chen et al., Proc Natl Acad Sci USA 90:6508-12 (1993)) and six histidine residues (His tag) for detection and purification with anti-HA and/or Ni-NTA.

[0082] To purify sufficient Fab for ELISA based assays and in vitro neutralization tests in a higher throughput format, nickel-NTA column chromatography (Qiagen) is used. In this case, Fabs that have been subcloned (either before or after screening) to include a His tag are grown in 1 liter of SB to an OD₆₀₀ of 0.8 and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3-4 hours at 30° C. to produce optimum amounts of Fab. To isolate Fab from the periplasmic space, cell pellets are resuspended in cold 1× PBS with added Complete Mini (Roche) protease inhibitor and are sonicated using a Sonics Vibra-cell VC750. Cellular debris is then pelleted and the supernatants are applied to Qiagen Ni-NTA columns. By using 16 of these columns, 75 μg of Fab per candidate was obtained in initial tests. By using a row of 12 columns per Fab in a single 96-well format, 8 Fabs can be purified, providing sufficient material for initial PRNT and ELISA assays. The epitope specificity tests require untagged Fab as well. These Fabs are purified on columns composed of goat anti-human F(ab′)₂ (Pierce) bound to Protein G or Protein A (Pharmacia) as described above in a 96 well format. Larger volumes of any desired Fabs can be purified by fast performance liquid chromatography (FPLC) (Pharmacia) on either the anti-human F(ab′)₂ column or on a nickel column. This method generally yields about 150-1000 μg of purified Fab/liter, though this varies from Fab to Fab.

[0083] Characterization of Purified Fabs.

[0084] Titration on Antigen.

[0085] Purified Fabs are titered against antigen in ELISA assays to compare the antigen-binding characteristics of Fabs within related groups established by sequencing.

[0086] Assays to Determine Epitope Specificity.

[0087] Epitope specificity can be determined by ELISA sandwich assays or by competition assays. Competition between Fab fused to gene III (fusion Fab, with or without phage attached) or a tag and purified Fab lacking gene III or a tag can be performed to assess epitope specificity. 50 μl of antigen at 4 μg/ml in PBS is incubated overnight at 4° C. in microtiter wells. After washing with PBS, wells are blocked with BC containing 1% Tween 20 in PBS at room temperature for 30 minutes. 50 μl PBS containing dilutions of one purified Fab are added to blocked wells and allowed to incubate at 37° C. for 1 hour. To this is added 50 μl of supernatant containing the second Fab as a fusion, and incubation proceeds for another hour at 37° C. The second Fab is detected with horse radish peroxidase-conjugated anti-M13 antibody (Pharmacia). Wells are developed with an HRP substrate buffer from Sigma, using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) tablets in a phosphate citrate buffer, pH 5.0. To use Fabs bearing the HA/His tag in this assay, the anti-M13 antibody used for detection above is replaced by either an alkaline phosphatase labeled anti-HA or labeled anti-His antibody detected with a PNPP assay.

[0088] Production and Purification of IgG from Fabs Identified as Neutralizing

[0089] Fabs are tested for their ability to neutralize the individual diseases using techniques known to those skilled in the art.

[0090] Conversion of Fabs to Full-Length IgG and Generation of Stable Cell Lines.

[0091] Fabs are subcloned in a two step process into a mammalian expression vector that creates a full-length IgG1 heavy chain. This vector utilizes a glutamine synthetase gene as a selectable marker, permitting growth of transfected cells in glutamine-free medium (Bebbington et al., Biotechnology 10:69-75. 1992). Vectors are transfected by electroporation using standard methods into the NSO mouse myeloma cell line. Stable cell lines are selected in glutamine-free medium and are isolated by limiting dilution. Pooled transfections can also be performed with this vector in NSO or CHO-K1 cells in order to examine smaller quantities of IgG prior to selecting a stable cell line. DNA prepared from each clonal line is analyzed by restriction digestion to determine successful insertion of the vectored immunoglobulin. Western blot analysis of media from each clonal line is used to assess production of full-length IgG, and a quantitative ELISA assembly assay is performed by capturing light chains and detecting heavy chains with appropriate antibody.

[0092] For purification of IgG, transiently infected cells or stable cell lines expressing IgG candidates are grown in miniPerm bioreactors (Vivascience) or in hollow fiber bioreactors. Supernatants are purified by FPLC using a protein G or protein A column. Additional purification can be accomplished using a hydrophobic interaction column.

[0093] In vitro and in vivo Testing of IgG

[0094] IgG derived from Fabs can be tested in vitro and in vivo in assays specific for the individual diseases as described below.

[0095] The above techniques have been successfully used in the cases of anthrax and VEEV. The same libraries and/or libraries created from additional human donors can be panned against Dengue Virus, WNV, and Vaccinia Virus. The same techniques for converting Fabs to whole IgG and IgG purification can be used.

[0096] The present antibodies or antibody fragments may be used in conjunction with, or attached to other antibodies (or parts thereof) such as human or humanized monoclonal antibodies. These other antibodies may be catalytic antibodies and/or reactive with other markers (epitopes) characteristic for a disease against which the antibodies are directed or may have different specificities. The antibodies (or parts thereof) may be administered with such antibodies (or parts thereof) as separately administered compositions or as a single composition with the two agents linked by conventional chemical or by molecular biological methods. Additionally the diagnostic and therapeutic value of the antibodies may be augmented by labeling the antibodies with labels that produce a detectable signal (either in vitro or in vivo) or with a label having a therapeutic property.

[0097] The antibodies in accordance with this disclosure and/or fragments thereof can be used in a variety of in vitro and in vivo immunoassays to detect the presence of an infective agent in a subject or to detect the presence of antibodies produced by a subject in response to exposure to an infective agent. Suitable immunoassays include, by way of example, radioimmunoassays, both solid and liquid phase, fluorescence-linked assays or enzyme-linked immunosorbent assays or assays based on fluorescence resonance energy transfer (FRET) technology.

[0098] In one embodiment, an ELISA assay can be used to detect the presence of human antibodies against toxins in patient fluids. In a typical assay procedure, an antigen associated with an infective agent, such as antigen PA83 (List Laboratories) is placed in solution and bound to Immulon 2 HB plates (VWR) and allowed to incubate overnight, preferably at about 4° C. Wells are then washed and blocked by incubation for about one hour with a mixture of a suitable PBS derived solution and Tween 20. Wells are washed and allowed to incubate for about one to about two hours at about 37° C. with patient samples (for example blood, serum, pleural lavage fluids) either straight or as a dilution series. As positive controls and for quantitation, some wells are incubated with the antibodies against an infective agent produced in accordance with the present disclosure. Wells are washed and then incubated for about one to about two hours at about 37° C. with a secondary antibody, which may be alkaline-phosphatase labeled goat anti-human F(ab′)₂. Wells are washed again and then detected using commercially available means and ELISA readers.

[0099] Variations on this assay include binding an antigen associated with an infective agent, such as the PA83 antigen, to other solid supports, such as dipsticks or beads, identification using other secondary antibodies, such as goat anti-human IgG, and detection using alternate labels, such as horseradish peroxidase detected with the Turbo TMB-ELISA kit (Pierce).

[0100] In another embodiment, two antibodies against an antigen associated with an infective agent, such as PA83, are used to detect and quantitate the amount of antigen in a sample. The first antibody is bound to a solid substrate (for example, a microtiter plate, beads, or a dipstick). For example, an antibody produced in accordance with the present disclosure may be placed in solution and incubated overnight at about 4° C. in a microtiter well. Wells are then washed and blocked by incubation for 1 hour with a mixture of a suitable PBS derived solution and Tween 20. Wells are washed and allowed to incubate for about one to about two hours at about 37° C. with patient samples (for example blood, serum, pleural lavage fluids) either straight or as a dilution series. A standard curve using a dilution series of antigen is also included. Wells are then washed and incubated for about one to about two hours at about 37° C. with a second anti-anthrax antibody that binds a non-competitive epitope on the antigen. This second antibody is labeled with alkaline phosphatase. Wells are washed and then detected using commercially available means and ELISA readers.

[0101] Variations on this assay include binding the first antibody to other solid supports, utilizing different concentrations of antibody and binding conditions and methods of stabilizing the support/antibody binding for use in commercial assays, blocking or washing with alternate solutions, using different labels on the second antibody or alternate detection systems, or using an unlabeled second antibody following with a third labeled antibody to detect the second. It also includes variations where only the first or second antibody is a human antibody, and the other is an antibody from another entity or from another animal source.

[0102] In another embodiment, an immunoassay utilizes at least one anti-infective agent monoclonal antibody and at least one labeled analyte, which can be a labeled antibody or a labeled peptide, preferably an anti-infective agent antibody, and most preferably, a polyclonal antibody, in a sandwich immunoassay comprising:

[0103] a) coating a solid phase with the anti-infective agent monoclonal antibody,

[0104] b) adding a test sample to the coated solid phase and incubating the two,

[0105] c) washing the solid phase,

[0106] d) adding labeled anti-infective agent antibody and incubating the same,

[0107] e) washing the solid phase, and

[0108] f) detecting label activity to determine the presence of the infective agent.

[0109] The labeled antibody may have binding specificity for the antibody on the solid phase or the infective agent. The wash solution is generally a buffered solution, but may be water or may contain other components.

[0110] The test sample is a body fluid or tissue obtained from the body of an animal and is preferably plasma, but other body fluids such as serum, whole blood, urine, cerebral spinal fluid and synovial fluid may be used. The label may be an enzyme known to those skilled in the art such as horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, luciferase and beta-galactosidase. Examples of non-enzyme labels include fluorescent labels, such as fluoroisothiocyanate, rhodamine or fluorescein, radioisotopes for radioimmunoassays, and particles.

[0111] In another embodiment, an immunoassay is performed using Fluorescence Resonance Energy Transfer (FRET) technology. As one example of this, an antibody against an antigen of an infective agent is labeled with one chromophore while a second antibody against another epitope on the same antigen is labeled with an alternative chromophore. Either or both of these antibodies can be produced in accordance with the present disclosure. The chromophores are chosen such that when placed in extremely close proximity, such as by binding to the same antigens, they interact so as to produce a fluorescent signal. Thus, addition of these two antibodies to a patient sample for dilution thereof will produce a detectable fluorescent signal in the presence of the appropriate antigen of the infective agent.

[0112] Other methods for the in vitro detection of infective agents, which are provided as examples but are not intended to be limiting, include competitive inhibition assays, single step assays, and agglutination assays.

[0113] The presence of elevated levels of the antibody or antibody fragment in the sample correlates with the presence of the infective agent and disease caused thereby in the subject. Where the assay is for an antibody to the infective agent, elevated levels of a secondary antibody or antibody fragment to the antibody to the infective agent correlates with the presence of the infective agent and disease caused thereby in the subject.

[0114] The present disclosure includes diagnostic test kits to be used in assaying for infective agents or antibodies thereto in samples, comprising at least one anti-infective agent monoclonal antibody. In addition, diagnostic kits may contain buffer solutions, labeled polyclonal or monoclonal anti-infective agent antibodies, antigens or peptides and any accessories necessary for the use of the kit.

[0115] In another aspect, the present disclosure provides vaccines for prophylactic treatment against infection by anthrax virus. These vaccines include a multimer of PA63 in a pharmaceutically acceptable carrier. The multimer of PA63 can contain up to twelve PA63 units. The multimer may thus be a dimer, trimer, quadrimer, pentamer, hexamer, heptamer, octamer, etc. In particularly useful embodiments, the multimer of PA63 contains up to seven PA63 units, with a heptamer of PA63 being preferred. The vaccine can be administered prophylactically to a subject in advance of exposure to anthrax virus.

[0116] The present antibodies or antibody fragments herein may typically be administered to a patient in a composition comprising a pharmaceutical carrier. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in the carrier. Pharmaceutically accepted adjuvants (buffering agents, dispersing agent) may also be incorporated into the pharmaceutical composition. It should be understood that compositions can contain both entire antibodies and antibody fragments.

[0117] The antibody and/or fragment compositions may be administered to a patient in a variety of ways. Preferably, the pharmaceutical compositions may be administered parenterally, e.g., subcutaneously, intramuscularly, epidurally or intravenously. Thus, compositions for parenteral administration may include a solution of the antibody, antibody fragment, or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody or antibody fragment in these formulations can vary widely, e.g., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

[0118] Actual methods for preparing parenterally administrable compositions and adjustments necessary for administration to subjects will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 17^(th) Ed., Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.

EXAMPLE 1 Anthrax

[0119] Phage libraries were developed from messenger RNA isolated from blood and bone marrow of active service military donors who had been vaccinated against anthrax. Blood samples were collected from military physician volunteer donors who received their AVA anthrax vaccine boost one week prior to collection. In addition, a commercial source supplied coded bone marrows with matched sera and some immunization records from active service military personnel. Several of the bone marrow donors and all of the blood donors had titer to anthrax antigen PA83 (FIG. 2). The bone marrow donor with the best titer against PA83 (951) had been immunized against anthrax three weeks prior to blood collection.

[0120] Total RNA was obtained from bone marrow samples 951 and 1037 and blood sample MD3 using Tri-reagent BD (Molecular Research Center, Inc.) according to the manufacturer's instructions. Messenger RNA was obtained using Oligotex (Qiagen) spin columns per manufacturer's instructions. Phage libraries expressing antibody Fab fragments (kappa or lambda light chains complexed to the variable and first constant regions of the heavy chain) were constructed in plasmid pAX243h vectors by proprietary methods as described in U.S. Provisional Application Nos. 60/287,355 and 60/323,455, the disclosures of which are incorporated herein in their entirety by this reference. Two Fab libraries were generated for each donor, one expressing kappa light chains, and one expressing lambda light chains, and all utilizing gamma heavy chains. Phage bearing Fabs from six libraries were panned through four rounds of enrichment against PA83. The 951 libraries were also separately panned through four rounds of enrichment against purified PA63, which was generated from PA83 as described by Miller et al. (Miller et al., 1999). To remove phage that bound to PA63 sites shared with PA83, soluble PA83 was first allowed to bind at 20 μg/ml to the phage for one hour at 37° C., after which the mixture was incubated with PA63 bound to microtiter plate wells.

[0121] Recombinant PA83 antigen was obtained from USAMRIID at Fort Detrick and was used in ELISA assays to identify anthrax-vaccinated individuals from the armed forces who have the highest titers against the PA83 antigen. RNA has been isolated from the bone marrow or blood of these individuals, and a Restriction Enzyme Digestion/Nested Oligonucleotide Extension Reaction/Single Primer Amplification (RED/NOER/SPA) was used to obtain combinatorial Fab libraries from this RNA. See FIG. 2.

[0122] RNA from the three highest titer individuals was used to construct libraries using the RED/NOER/SPA method of amplification. Two libraries, 951 and 1037, were from bone marrow donors received from Poietics (Menlo Park, Calif.). The third library, MD3, was from the blood of a vaccinated volunteer. The efficiency of the library ligations is shown in the following Table 1: TABLE 1 Library Light Chain Heavy Chain  951 Kappa  5.7 × 10⁹ 2.17 × 10⁹  951 Lambda  2.6 × 10⁹ 1.86 × 10⁹ 1037 Kappa 3.07 × 10⁹ 3.29 × 10⁹ 1037 Lambda 4.52 × 10⁹ 1.07 × 10⁹ MD3 Kappa 6.65 × 10⁸  2.6 × 10⁸ MD3 Lambda  8.7 × 10⁸  4.2 × 10⁸

[0123] All of the libraries were panned against PA83, and the 951 libraries were panned against PA63. For PA83, antigen was bound to wells and blocked prior to addition of phage bearing the displayed Fab fragments. For PA63, the display phage were initially mixed with PA83 before being reacted with PA63 antigen bound to wells, in order to diminish the recovery of phage that reacted to antigens shared by both PA83 and PA63. PA63 was generated and purified from PA83 following the method described by Miller et al. (1 999). The results for panning of two of the libraries and the panning against PA63 are shown in the following table.

[0124] Panning was performed, initially with the 951 and MD3 libraries against PA83, and with the 951 libraries on PA63. ER2738 cells were used, aside from the initial library transformations into XL1-Blue. Input, output, and some initial ELISA results for both panning rounds are shown in the following tables. TABLE 2 Panning against PA83 and PA63 Input titer Output titer % ELISA Library Round Antigen (total) (total) positive 951 K 1 PA83 5.9 × 10¹⁰ 1.2 × 10⁵ 2 PA83 1.1 × 10¹⁰ 3.5 × 10⁵ 10% 3 PA83 9.1 × 10¹⁰ 1.2 × 10⁶ 96% 4 PA83 4.5 × 10¹⁰ 1.0 × 10⁸ 96% 951 K 1 PA63 3.9 × 10¹⁰ 1.9 × 10⁵ 2 PA63 5.0 × 10⁹  9.6 × 10⁵ 3 PA63 5.0 × 10¹⁰ 7.6 × 10⁴ 4 PA63 2.5 × 10¹⁰ 1.5 × 10⁵ 15% 951 L 1 PA83 8.8 × 10¹⁰ 7.5 × 10⁴ 2 PA83 2.1 × 10¹⁰ 7.3 × 10⁵  4% 3 PA83 9.1 × 10¹⁰ 1.7 × 10⁶ 68% 4 PA83 8.2 × 10¹⁰ 1.6 × 10⁸ 89% 951 L 1 PA63 5.9 × 10¹⁰ 2.3 × 10⁵ 2 PA63 5.6 × 10⁹  8.6 × 10⁵ 3 PA63 5.4 × 10¹⁰ 4.8 × 10⁴ 4 PA63 3.3 × 10¹⁰ 1.5 × 10⁵ 15-30% MD3 K 1 PA83 1.3 × 10¹¹ 1.9 × 10⁴ 2 PA83 6.9 × 10¹⁰ >10⁷ 47% 3 PA83 3.9 × 10¹⁰ 3.5 × 10⁷ 70% 4 PA83 9.3 × 10¹⁰ 1.1 × 10⁸ 75-89% MD3 L 1 PA83 4.8 × 10¹⁰ 8.8 × 10⁴ 2 PA83 5.7 × 10¹⁰ >10⁷ 21% 3 PA83 4.5 × 10¹⁰ 8.3 × 10⁷ 40% 4 PA83 5.0 × 10¹⁰ 1.7 × 10⁸ 40%

[0125] Enrichment is evident for all the PA83 panned libraries. Libraries panned against PA63 showed some candidates with very weak reactivity to PA83. These candidates were positive when tested against PA63. Sequence analysis of the VH and VK or VL regions of positive responders is indicated in FIGS. 3-5. Though certain sequences predominate, diversity can be demonstrated. The Fabs that were panned against PA63 with PA83 preabsorption appear to contain significantly different groups of sequences than those that were panned against PA83.

[0126] After panning, individual candidates from various panning rounds of all four PA83-panned libraries were screened for reactivity to PA83 by ELISA. In order to identify PA63 binding Fab fragments, 951 kappa and lambda library phage that had been panned against PA63 were first screened for binding to PA83, initially to eliminate PA83 binders from the screen. However, no candidates were found that bound PA83 well, indicating that the competition provided by incubating the phage initially with soluble PA83 was effective. A small percentage of clones in the fourth round panning of both the anti-PA63 libraries showed very weak ELISA reactivity after several hours of incubation in substrate. These clones were screened against PA63 resulting in a much stronger signal. The weak reactivity to PA83 may be due to cross-reactivity with PA83, or may reflect a small amount of PA63 in the PA83 preparation, which might have resulted from protease cleavage of PA83 at the furin protease sensitive site (Klimpel et al., 1992) during purification or storage.

[0127] Over 144 individual candidates with strong PA83 or PA63 binding activity selected from the six different panned libraries were sequenced and a panel of all variant candidates was identified. This included thirty-one unique PA83 binders and six unique PA63 binders. Twenty-five of the unique PA83 binders were all derived from variable heavy chain (VH) locus 3-30/3-30.5. Among the heavy chains of the PA63 binders, two related sequences were predominantly seen; these were dissimilar to the PA83 sequences. Because a single mutation can dramatically alter the affinity of an antibody, candidates were considered unique if they had one amino acid difference in either their heavy or light chains as compared to other candidates.

[0128] Additional antibody sequences to the anthrax proteins PA83 and PA63 are presented in FIGS. 6-8C. For the human kappa light chain variable sequences shown in FIG. 6, the first two amino acids, S (serine) and R (argentine) are derived from the Xba I (TCTAGA) site used in cloning. Amino acid number three in the figure corresponds to amino acid number one for human kappa light chains in the Kabat numbering system (Sequences of Proteins of Immunological Interest, Kabat et al., 1991). The last four amino acids (RTVA) indicated for most of the sequences corresponds to the first four amino acids of the human kappa light chain constant regions, numbered 108-111 in the Kabat numbering system. Two sequences shown do not quite extend to the beginning of the constant region. Because the variable region includes length polymorphisms, the actual number of amino acids in each sequence may be larger or smaller than 113 (the two initial amino acids, plus 111). For the human lambda light chain variable sequences shown in FIG. 7, the first two amino acids, S (serine) and R (argentine) are derived from the Xba I (TCTAGA) site used in cloning. Amino acid number three in the figure corresponds to amino acid number one for human lambda light chains in the Kabat numbering system. The last amino acid indicated for each sequence corresponds to amino acid 155 of the human lambda light chain constant regions in the Kabat numbering system. Because the variable region includes length polymorphisms, the actual number of amino acids in each sequence may be larger or smaller than 157. For the human gamma heavy chain variable sequences shown in FIGS. 8A-C, the first two amino acids, L (leucine) and E (glutamate) are derived from the Xho I (CTCGAG) site used in cloning. Amino acid number three in the figure corresponds to amino acid number one for human gamma heavy chains in the Kabat numbering system. The last amino acid indicated for each sequence corresponds to amino acid 118 of the human gamma heavy chain constant regions in the Kabat numbering system. Because the variable region includes length polymorphisms, the actual number of amino acids in each sequence may be larger than 120.

[0129] Panning against EF and LF, which are also present in small amounts in the AVA vaccine used to immunize military personnel, is performed with the present libraries. Additional panning against PA63 can be performed with the other libraries. Biacore assays are done to assess affinity of the different antibodies. Competition experiments are performed to identify groups of antibodies that share the same epitope binding characteristics. Candidates are assessed for their ability to block the binding of PA with either receptor, EF or LF in cellular assays. The best candidates are then tested for their ability to block toxicity in vivo in animal models, either using PA, EF and LF or actual anthrax infection. Candidates are optionally converted to full length human antibodies one or more of these tests.

[0130] To generate purified Fab for additional testing, candidates from this panel underwent a subcloning step to remove gene III from the heavy chain portion of the Fab fragment. Fab was then purified from two to four liters of culture by fast performance liquid chromatography (FPLC) using a goat anti-human Fab column. Neutralization assays using the purified Fab were performed using a mouse macrophage cell line, J774A.1, after the manner of Little et al. (Little et al., 1990). Conditions were established for using the Cytotox96 detection kit (Promega) to assay lactate dehydrogenase (LDH) released by cell death in response to toxin action. J774A.1 cells were plated overnight at 14,000 cells/well in 96 well dishes. 4-8 wells were assayed for each point. Fabs were used at 50 nM. Toxin was generated as follows: PA83 was added at 400 ng/ml (4.6 nM), with LF at 40 ng/ml. After incubation at 37° C. for 4 hours, wells were examined microscopically and then media was removed and centrifuged to pellet unattached cells.

[0131] Results of a number of neutralization assays are summarized in FIG. 9. These Fabs include F9L6R2 (also referred to herein as 951L6R2 and 83L6R), FML5B (also referred to herein as 83L5B), FMK9C (also referred to herein as 83K9C), F9K3C (also referred to herein as 83K3C), F9K2A (also referred to herein as 83K2A), FML8E (also referred to herein as 83L8E), FML8F (also referred to herein as 83L8F), FML3B (also referred to herein as 83L3B), FML2D (also referred to herein as 83L2D), FML7D (also referred to herein as 83L7D), F9K3H (also referred to herein as 83K3H), FML4E (also referred to herein as 83L4E), FML2E (also referred to herein as 83L2E), F9K2H (also referred to herein as 83K2H), F9K7H (also referred to herein as 83K7H), FMK7C (also referred to herein as 83K7C), and 951L631D (also referred to herein as 63L1D). As can be seen, fourteen of the seventeen anti-PA Fabs (samples e-u) tested are able to neutralize the effects of the anthrax toxin with greater than 80% viability. Five Fabs neutralize fully at this concentration and in this time frame. Samples (a) and (b) are two of the Fabs without the addition of toxin; these demonstrate that cell death is not caused in this time period by endotoxin in the purified samples. Sample (c) shows the effect of toxin alone. Sample (d) contains an irrelevant Fab that does not protect cells significantly from the action of the anthrax toxin.

[0132] Selected Fabs were titrated to determine their 50% protection values in vitro (FIG. 10). Fabs were serially diluted and aliquots were added to media containing toxin. In these experiments, PA was at a final concentration of 400 ng/ml, and LF at 80 ng/ml. These aliquots were then added to cells in quadruplicate and incubated at 37° C. for 4 hours. Cytotoxicity was assessed visually and was quantitatively measured with the Cytotox96 assay as described. The anti-PA83 Fabs shown here all have 50% neutralization values that are close to equimolar with the concentration of PA83 used in the assay. The anti-PA63 Fab 951L631D, however, has a 50% neutralization value that is about 5-7 fold lower than these; in other words, one molecule of 951L631D neutralizes many molecules of PA83. PA83 is cleaved and converted by the J774A.1 cells in this experiment to heptameric pores. The most probable explanation for the ability of 951L631D to neutralize substoichiometric amounts of PA83 is that it is acting at the level of the heptameric pore, and is effectively neutralizing up to seven PA83 molecules at once.

[0133] 951L631D and MK7C have recently been tested in vivo. Two rats receiving 40 pg of PA83 and 8 μg of LF in 200 μl total volume of PBS died in 60 and 71 minutes. Two rats receiving the same quantities of toxin and 310 μg of 951L631D survived for 25 hours, at which time they were sacrificed. At about 3-5 hours, these rats showed some symptoms of illness, such as lethargy and a slight panting, but at 16 hours this had disappeared in one rat, while the other remained lethargic but had normal breathing. By 25 hours both rats appeared normal as compared to the PBS injected control rat. 951L631D therefore appears capable of protecting rats against anthrax intoxication in vivo. MK7C has been tested at 300 μg with toxins in one rat which survived without showing any symptoms.

[0134] Fabs generated from 9K2H (also referred to herein as 83K2H), 9L6R2 (also referred to herein as 951L6R2 and 83L6R), MK7C (also referred to herein as 83K7C), 9K7H (also referred to herein as 83K7H), ML8E (also referred to herein as 83L8E), and 951L631D (also referred to herein as 63L1D) were tested for their ability to react with linear epitopes. PA83 and PA63 were run under denaturing (but non-reduced) conditions in an SDS-PAGE gel and transferred to nitrocellulose filters by Western blofting. Strips cut from the blots containing either PA63 or PA83 were hybridized to each of these purified antibodies overnight at the same concentrations. Bound antibody was reacted with alkaline phosphatase conjugated goat anti-human F(ab′)₂ (Pierce), and the results are shown in FIG. 11. Fab 63L1D did not bind monomeric PA63 or PA83. This data shows that Fab 63L1D binds to a conformational epitope. All of the anti-PA83 Fabs used bound PA83 well under these conditions, demonstrating binding to a linear epitope or one which might be reformed during Western transfer conditions. 83K7C bound equally well to PA83 and PA63 as seen before. 83L8E and 83L6R showed some binding in the Western to PA63. This may be because the amounts of Fab and antigen used were high, or because PA63 is monomeric on the Western, whereas in the ELISA of FIG. 14 conditions were such that it was mostly heptameric.

[0135] All of the five anti-PA83 Fabs tested appear to bind to linear epitopes on PA83 (FIG. 12). The anti-PA63 antibody, in contrast does not bind to denatured PA63, and shows what appears to be faint, presumably non-specific binding to PA83. 9K2H and 9K7H show no binding to denatured PA63, whereas MK7C and ML8E bind strongly, with 9L6R2 showing weak binding.

[0136] The ability of some of these Fabs to bind to PA83 and PA63 was further analyzed quantitatively by performing a Fab titration against antigen in an ELISA format. PA83 and PA63 were purchased from List Laboratories and resuspended in water or 50% glycerol, respectively, per instructions. The graph below shows the titration of four Fab fragments against PA83 or PA63. Closed symbols represent reactivity to PA83, open symbols to PA63. The results show that Fab 63L1D binds to PA63, but not to PA83, indicating that it binds to an epitope that is only available after conversion of PA83 to PA63. Fab 83K7C binds to both PA63 and PA83 in ELISA, whereas two other Fabs originally selected on PA83 have significantly decreased binding to PA63 as compared to PA83. Note that the saturation value reached by Fab 63L1D against PA63 was only about one-fourth that of Fab 83K7C. These observations demonstrate Fab 63L1D binding to a conformational epitope formed by the heptamerization of PA63. Lowered binding in ELISA was due to a more limited number of available sites for binding on a heptamer, and reflects less of the PA63 being properly heptamerized and available for binding. Because PA83 binds equally well, the absolute quantity of PA63 available was reasonably equivalent. LF is known to bind to a conformational epitope formed by the heptamer (Cunningham et al., 2002; Mogridge et al., 2002); though present in seven places, LF is only capable of binding on three, because of steric hindrance of the bound LF.

[0137] In FIG. 13, a his tagged version of Fab FML8E was generated and used in competition with other untagged Fabs to assess epitope specificity. Fabs F9K2H, F9K7H, and FML8F all compete similarly to self-competition with FML8E, suggesting that these Fabs recognize the same epitopes, or epitopes in close proximity to that seen by FML8E. F951L6R2 competes, but not as well, suggesting that this epitope is not the same, though it may be close enough to cause competition. FMK7C is very ineffectual in competition, indicating that it probably binds at a distant site. Interestingly, cleavage to PA63 abolishes binding by F9K2H and F9K7H, as shown in the Western blot figure above, while binding on the Western is still evident for FML8E and F951L6R2, and some reactivity is also seen at high concentrations in the ELISA titration above. This suggests that the epitopes for binding F9K2H/F9K7H are not the same as for FML8E/FML8F, F951L6R2, or FMK7C. Fabs indicated were serially diluted 1:4 and bound for one hour at 37° C. to microtiter wells that had been coated overnight with 200 ng of PA83. His-tagged FML8E was then added without washing at 5 pg/ml and allowed to react for 2 hours, after which plates were washed and reacted with alkaline phosphatase conjugated anti-His for a PNPP assay. Note that FML8E and FML8F have similar heavy chains, but different light chains. F9K2H and F9K7H are related to each other and use the same heavy chain germline locus as FML8E, but have quite different CDR regions from ML8E. F951L631D and FMK7C are from different heavy chain germline loci.

[0138] The ability of additional anti-PA83 Fabs to bind to PA63 in ELISA was assessed at a concentration of 1 μg/ml against PA63 or PA83 at 200 ng/well. A concentration of 1 μg/ml was used because this was the concentration as set forth in FIG. 12 where binding of 83L8E and 83L6R was seen to be either absent or reduced. As seen in FIG. 14, the only anti-PA83 Fab that bound PA63 appreciably was 83K7C.

[0139] Because it appeared as if Fab 63L1D might be binding in a manner similar to LF, an experiment was performed to determine whether Fab 63L1D could compete with LF for binding to PA63 in ELISA. For this assay, PA63 was bound to microtiter-well dishes at 200 ng/well. Wells were washed and blocked and LF was serially diluted as indicated and incubated in quadruplicate for 2 hours at 37° C. Fab 63L1D and 83K7C were used at a final concentration of 1 μg/ml, which is a concentration somewhat higher than the 50% maximum binding concentration identified by ELISA titration for each Fab against PA63. Fabs were added to wells and allowed to compete for 15 minutes and 2 hours. Bound Fab was detected with alkaline phosphatase labeled goat anti-human Fab and a PNPP assay.

[0140] The results of this experiment are shown in FIG. 15. 83K7C was seen to bind equally well at all concentrations of LF, indicating that it did not compete with LF for binding to PA63. 63L1D binding for 15 minutes showed a decrease at higher concentrations of LF, indicating that it did compete with LF for binding to PA63. When competition was allowed to continue for two hours, more 63L1D was able to bind, even at the highest concentrations of LF. This data shows that Fab 63L1D could compete away LF.

[0141] A competitive ELISA was performed in which a mouse monoclonal antibody (14B7) was mixed with different concentrations of either Fab 83K7C or 83L8E and then allowed to bind to PA83 immobilized on a microtiter plate. Mouse monoclonal antibody 14B7 was obtained from Stephen Leppla (Little et al., 1988). This monoclonal antibody has been shown to bind PA83 and to block the binding of PA83 to its cellular receptor (Little et al., 1996). Bound 14B7 was detected using an alkaline-phosphatase conjugated goat anti-mouse IgG Fc. FIG. 16 shows that 83K7C, but not 83L8E, competes for binding with 14B7. Thus, Fab 83K7C binds to a similar or overlapping epitope, and acts by blocking receptor binding.

[0142] Kinetic analysis using Biacore (Biosensor Tools, Salt Lake City, Utah) surface plasmon resonance (SPR) was conducted to determine kinetic and binding parameters for Fab/toxin interaction. Association (k_(a)) and dissociation (k_(d)) rate constants were measured by Biacore; K_(D) was calculated as (k_(d)/k_(a)). The number in parentheses represents the standard error in the last significant digit. Residual standard deviation represents on average the number of RUs each data point deviates from the model. The results, set forth in Table 3 below, show that 63L1D and 83K7C bind with subnanomolar affinity to immobilized PA63. TABLE 3 Res. Stand. Interaction k_(a) (M⁻¹ s⁻¹) k_(d) (s⁻¹) k_(D) (nM) Dev. Fab 83K7C/PA83 1.16(1) × 10⁵ 4.26(1) × 10⁻⁴ 3.67 0.597 Fab 83K7C/PA63 9.77(1) × 10⁴ 8.50(5) × 10⁻⁵ 0.87 0.71 Fab 63L1D/PA83 nd nd nd nd Fab 63L1D/PA63 1.50(4) × 10⁶ 1.90(1) × 10⁻⁴ 0.13 0.519

[0143] Fab 63L1D did not bind PA83, so no value was determined. This was consistent with the ELISA data showing that Fab binds to the PA63 heptamer or an epitope exposed following PA83 cleavage. Interestingly, 83K7C bound even more tightly to PA63 then to PA83, primarily due to a lowered off rate.

[0144] This disclosure demonstrates for the first time that human anti-anthrax toxin antibodies which possess a high affinity and are potently neutralizing in vitro, can be isolated from AVA immunized donors. Little et al. (1990) identified a panel of murine monoclonal antibodies against the anthrax toxin lethal factor. Evaluation of in vitro versus in vivo protection suggests that the degree of protection in vitro may correlate with protection in vivo, except for rare cases. Fifteen neutralizing antibodies have been identified from the nineteen examined, some of which neutralize fully at low concentrations. It is anticipated, therefore, that some of these antibodies will be protective in vivo. The data further suggests that the AVA vaccine is effective in protecting humans against anthrax exposure.

[0145] Both anti-PA83 and anti-PA63 activities in combination have potential for in vivo therapeutic purposes. Anti-PA83 would limit the number of PA83 molecules binding to cellular receptors. Those PA83 molecules that were not destroyed and did form heptameric pores would then be neutralized by anti-PA63 activity, providing potent protection against the lethal effects of an anthrax infection. The combination of the two antibodies could provide immediate protection against the formation of new functional pore structures either at the onset or during the course of an infection.

[0146] The use of these two antibodies could provide additional passive protection to personnel, vaccinated or unvaccinated, that might be exposed to a suspected anthrax release. The availability of a therapeutic that could protect in the face of disease would help to alleviate public anxiety about anthrax. In addition, such a therapeutic agent might make the deliberate release of anthrax less successful as an act of bioterrorism, and may therefore decrease the likelihood of such attacks.

[0147] In vitro Experiments

[0148] Conditions were established for using the Cytotox96 detection kit (Promega) to assay lactate dehydrogenase (LDH) released by cell death in response to toxin action. Mouse macrophage cell line J774A.1 (Little et al., 1990) was plated overnight at 14,000 cells/well in 96 well dishes. 4-8 wells were assayed for each point. Fabs were used as indicated in the figures. Toxin was generated as follows: PA83 was added at 400 ng/ml (4.6 nM) with LF at 80 ng/ml. After incubation at 37° C. for 4 hours, wells were examined microscopically and then media was removed and centrifuged to pellet unattached cells. Cytotox96 (Promega) assays of media were performed per manufacturer's instructions.

[0149] Selected Fabs were assayed for in vitro neutralization activity using serial dilution, and neutralization curves are given in FIG. 10. The anti-PA83 Fabs shown in FIG. 10, including 83K7C, all had 50% neutralization values that were close to equimolar with the concentration of PA83 used in the assay (4.6 nM). The anti-PA63 Fab 63L1D however, had a 50% neutralization value that was about 3.5 to 6 fold lower than the values obtained for the PA83 Fabs, which was substoichiometric with respect to PA83. This is again consistent with Fab 63L1D binding to a conformational epitope found on the heptamer, and therefore being able to effectively neutralize more than one PA molecule at a time. Both 63L1D and 83K7C fully protect cells from cell death at higher concentrations, a reproducible result.

[0150] To further characterize the binding of Fabs 63L1D and 83K7C, an additional experiment was performed to determine whether selected Fabs could neutralize the effect of toxin after PA had bound to cells. Those which act prior to the binding of LF to the heptamer would not be expected to block activity. Accordingly, PA83 was added to cells at a concentration of 400 ng/ml and allowed to incubate at 4° C. for 2 hours, after which cells were washed and LF at 80 ng/ml and Fab at 50 nM were added. The results of this assay are shown in FIG. 17. As can be seen, Fab 63L1D prevents cell death, consistent with the conclusion that it binds to the heptamer at a site that can prevent LF binding.

[0151] Animal Pharmacology Experiments

[0152] Animal procedures were approved by the Institutional Care and Use Committee at Perry Scientific, Inc., where experiments were performed. Fisher 344 rats were injected with 40 μg of PA and 8 μg of LF per 250 g rat as described in Ezzell et al. (1984), except that the tail vein was used. Toxin alone was used for the positive control; toxin with varying amounts of Fab was utilized as indicated for other groups. After initial trials on one or two rats indicated that Fabs 83K7C and 63L1D had a protective effect, a dose response study was performed for these two Fabs. Four rats were used per group. Negative controls were injected with PBS from Fab dialysis that was used as a vehicle. Surviving rats were sacrificed after 7 days.

[0153] The results of testing Fabs 83K7C and 63L1D in vivo against recombinant toxin challenge are shown in FIG. 18. As can be seen, 83K7C and 63L1D have different patterns of protection. 83K7C protected fully at both 2 and 6 nmoles (˜100 or 300 μg/rat, respectively), with a minimal but statistically significant delay of symptoms and death at 0.6 nmoles (p=0.0005 and p=0.038 respectively, two-tailed Student's t-test). 63L1D protected fully at 6 nmoles. At 2 nmoles, 63L1D protected from death, but animals began to show symptoms of anthrax intoxication (an altered respiration pattern) at about two and one-quarter hours after injection. Symptoms remained minimal for one or two hours and eventually subsided and animals survived. At 0.6 nmoles (˜30 μg/rat), 63L1D exhibited a substantial delay of symptoms and death. The difference in effect of these two Fabs is related to their modes of action. 83K7C binds PA83 and PA63 equally well in ELISA and acts to prevent binding of toxin to cells. As described above, 63L1D binds to the heptamer after its formation on cellular surfaces. Though 2 nmoles of 63L1D appear to be sufficient to protect rats for 2 hours in the presence of anthrax toxin, Fab is cleared more rapidly than LF from the animal. Symptoms then arise as remaining LF enters cells on previously bound PA, but the amount of unneutralized toxin generated is insufficient to cause pulmonary edema and secondary shock leading to death. Because clearance of Fab may in some instances contribute to the appearance of symptoms, use of a full-length IgG version of 63L1D will provide full protection at this or lower concentrations. At 0.6 nmoles, delay of symptoms and death is greater for 63L1D than for 83K7C. This result parallels the in vitro results, where 63L1D is more potent than anti-PA83 antibody fragments and can protect sub-stoichiometrically.

EXAMPLE 2 Venezuelan Equine Encephalitis Virus

[0154] Human Anti-VEEV Abs

[0155] The donor serums described above in connection with Example 1 were tested against TC-83 antigen using a standard ELISA assay (FIG. 19). Donors 1037, 811 and 951 had significant serum reactivity against TC-83. This indicated a high probability of obtaining anti-VEEV Fabs from antibody libraries made from the corresponding donor bone marrow. The IgG-kappa and IgG-lambda libraries for both 1037 and 951 (4 libraries in total) had previously been constructed for the anthrax example as described above. These phage-display antibody libraries were then panned through 4 rounds on TC-83 antigen. Results from this experiment are shown below:

[0156] Initial Library Sizes:  951K 5.7 × 10⁹  951L 2.6 × 10⁹ 1037K 3.1 × 10⁹ 1037L 4.5 × 10⁹

[0157] Round 1 Panning: Input Output  951K 5.6 × 10¹¹ 6.0 × 10⁴  951L 2.6 × 10¹¹ 1.8 × 10⁵ 1037K 4.8 × 10¹¹ 4.0 × 10⁴ 1037L 3.2 × 10¹¹ 1.0 × 10⁵

[0158] Round 2 Panning: Input Output  951K 1.2 × 10¹³ 1.2 × 10⁷  951L 3.0 × 10¹³ 1.0 × 10⁶ 1037K 3.8 × 10¹³ 4.4 × 10⁷ 1037L 2.2 × 10¹³ 4.8 × 10⁶

[0159] Round 3 Panning: Input Output  951K 5.0 × 10¹³ 3.1 × 10⁸  951L 7.8 × 10¹³ 4.0 × 10⁷ 1037K 8.0 × 10¹³ 6.1 × 10⁸ 1037L 8.8 × 10¹³ 2.1 × 10⁸

[0160] Round 4 Panning: Input Output  951K 1.0 × 10¹⁴ 8.0 × 10⁹  951L 4.4 × 10¹³ 2.0 × 10⁹ 1037K 1.5 × 10¹⁴ 1.0 × 10¹⁰ 1037L 3.8 × 10¹³ 4.0 × 10⁹

[0161] A panel of Fab clones from panning rounds 3 and 4 from all four libraries (951K. 951L, 1037K and 1037L) was screened for binding to immobilized TC-83 by ELISA using a Tecan robotic platform in a high throughput format. As seen in FIG. 20A-D, Fabs with significant binding to TC-83 (as detected using alkaline phosphatase conjugated anti-human Fab) were obtained in all 4 libraries. Fab clones were screened in comparison to positive control Hy4-26A (humanized variant of 3B4C-4) and a negative control anti-tetanus toxoid Fab which are the next to last and last samples respectively on each graph in FIG. 20. Three Fabs with the highest ELISA signals from each of the four libraries were selected for further analysis. DNA was prepared for each clone and then submitted for sequence analysis. The sequencing results showed that all three 951K clones were identical. Additionally, 10 of the 12 clones had the same variable heavy chain region (VH) but the majority of those Fabs had different light chain sequences. In all, there are 10 unique clones in 3 separate heavy chain (HC) groupings.

[0162] Four human Fab clones were chosen for further analysis. The selected Fabs represented all three distinct HC classes identified. VH classification Clone (generalized grouping) LC P3F2 #1 kappa P3F5 #2 kappa P3H6 #3 kappa P3G1 #1 lambda

[0163] All of the Fabs were purified from bacterial periplasmic preps using an anti-human F(ab′)₂ column on an FPLC. Because the P3H6 Fab had very low yield it was not pursued further.

[0164]FIG. 21 shows the binding activities of the three human anti-VEEV Fabs in a titration ELISA assay against TC-83. The purified anti-VEEV Fabs were also tested in a competition ELISA experiment, using the mHy4 Fab as the competitor. The results from this experiment are shown in the FIG. 22 and demonstrate the three VEEV Fabs do not compete for the same epitope (E2c) as the mHy4 Fab.

[0165] The human Fabs were not competitive for the E2^(c) epitope, but they may bind to other neutralizing epitopes on VEEV. To test that, an aliquot of each purified VEEV Fab was sent to collaborators at the CDC for use in a cell-based VEEV neutralization assay. The results from two separate experiments showed that P3F5 had very good neutralization capacity, similar to that seen with the positive control 3B4C-4. P3G1 also showed significant neutralization, while the P3F2 Fab had no apparent effect in the neutralization tests. TABLE 4 Fab/Ab Sample type Crosslinked? 70% PRNT 3B4C-4mAb positive control no (already bivalent) 25 ng/ml TT negative control yes >2500 ng/ml P3F2 Test yes >10,000 ng/ml P3F5 test yes 19.5 ng/ml P3F5 test no <78 ng/ml P3G1 test yes 156 ng/ml P3G1 test no 156 ng/ml

[0166] Table 4 reports the results of in vitro neutralization assay for VEEV. The titer of Ab or Fab required to give 70% reduction of VEE viral plaques in Vero cells is reported. The murine Ab 3B4C-4 (as whole IgG) was used as a positive control. Previously, bivalent antibody has been shown to neutralize virus more effectively, therefore anti-Fab cross-linking Ab was added to some wells (non-optimized concentration). A non-binding negative control Fab did not show neutralization at any concentration tested. Samples P3F5 showed activity near that of the murine 3B4C-4.

[0167] These preliminary results demonstrate that a fully human neutralizing anti-VEEV antibodies had been isolated. FIGS. 23A and 23B show the sequences for fully-human Fabs produced in accordance with this disclosure that neutralize VEEV. These existing human anti-VEEV Fabs can be converted to whole IgG as described above and purified for further characterization.

[0168] Test epitope specificity of the antibody for VEEV (Roehrig, et al Virology (1982) 118, pp269-278; and Roehrig and Mathews, Virology (1985) 142, pp 347-356).

[0169] Western Blot is run to see which of the TC-83 viral proteins is recognized by the Fabs. For Fabs that do not react by Western Blot, because a conformational rather than a linear epitope is recognized, native E1 and E2 envelope glycoproteins can be purified from viral lysate for ELISA or Radiolabeled immunoprecipitation assays as described previously.

[0170] Identification of the reactive epitope on the viral protein can be mapped using a competition ELISA with representative monoclonal antibodies for each binding group as listed below in Table 5. Microtiter wells coated with whole virus are incubated with an amount of the representative Ab that gives approximately 80% maximal binding. Wells also contain increasing amounts of the test Fab. Binding of the representative Ab to virus is monitored using an anti-mouse IgG Fc specific—Alkaline Phosphatase conjugate. Loss of binding is interpreted as competative binding by the test human Fab, indicating epitope specificity or spatial arrangement. TABLE 5 Representative Antibody Epitope 5B4D-6 gp56^(a) 2A4B-12 gp56^(b) 3B4C-4 gp56^(c) 1A6C-3 gp56^(d) 1A3A-5 gp56^(e) 1A4D-1 gp56^(f) 1A3A-9 gp56^(g) 1A3B-7 gp56^(h) 3B2D-5 gp50^(a) 3B2A-9 gp50^(b) 5B6A-6 gp50^(c) 3A5B-1 gp50^(d)

[0171] The representative Abs (from John Roehrig at the CDC, Ft. Collins, Colo.) can be obtained from ascitic fluid following a 50% ammonium sulfate precipitation and chromatography over a protein G column. Alternatively, Abs can be purified from the conditioned media of their hybridoma cell lines grown in Ig free media.

[0172] Test viral strain cross reactivity (Roehrig et al., J. Clin. Microbiology (1997) 35, pp1887-1890: and Roehrig et al., Virology (1982) 118, pp269-278).

[0173] VEEV is composed of six subtypes (1-6) with subtype 1 having five variants (1AB, 1C, 1D, 1E, and 1F). Virus strains from each subtype is tested by ELISA or indirect fluorescent antibody assay (IFA) as described previously for reactivity with each candidate Fab. Prototype viruses useful in these analysis are listed below in Table 6. TABLE 6 Strain Subtype TC-83 1AB Trinidad Donkey (TRD) 1AB P676 1C 3880 1D Mena 2 1E 78V-3531 1F Everglades (Fe3-7c) 2 Mucambo (BeAn 8) 3 Pixuna (BeAr 35645) 4 Cabassou (CaAr 508) 5 Ag80-663 6 Western Equine Encephalitis (WEE) (McMillan) Eastern Equine Encephalitis (EEE) (82V- 2137) St. Louis Encephalitis (SLE) (MSI-7)

[0174] Viruses from stocks maintained at the Division of Vector Borne Viral Diseases, Centers for Disease Control, Fort Collins, Colo., can be grown in BHK21 cells.

[0175] Perform in vitro Neutralization Test with Whole IgG.

[0176] Neutralization tests are done using 50-100 PFU/test in Vero cells, with 70% endpoints recorded as described previously (Roehrig et. al., 1982).

[0177] Test Ability of Abs to Protect Mice from Viral Challenge.

[0178] Known quantities of purified IgG diluted in PBS are inoculated i.v. via a tail vein into young mice, such as 3 week old NIH Swiss mice. Twenty-four hours later, mice are challenged i.p. with VEEV diluted in cell culture media. Controls receive PBS i.v. and either virus or virus diluent. An additional control group includes murine Ab 1A4A-1 or 3B4C-4 previously shown to provide protection. Mice are observed for 2 weeks. Heparinized plasma specimens from inoculated mice are obtained by bleeding from the reto-ocular venous plexus.

[0179] Isolation of Additional Anti-VEEV Fabs

[0180] An extended panel of human Fabs directed against TC-83 antigen is generated. Additional ELISA screens of >1000 individual Fab clones from 1037 and 951 libraries which have already been panned on TC-83 are performed. This supplements the original screen of 190 Fab clones from those panned libraries. In addition, new phage display antibody libraries are constructed from the RNA of a donor (811) previously shown to have titer against TC-83. The newly constructed 811 libraries are panned on immobilized TC-83. Unique Fabs are characterized as described above for their ability to provide neutralization in vitro and protection in animal models against lethal viral challenge.

EXAMPLE 3 Botulinum

[0181] By applying the previously described library creation and panning technologies, antibodies that bind many of the different botulinum toxin serotypes are isolated and produced in large quantities. As with the neutralizing antibodies described above for anthrax and VEEV, these fully human antibodies against botulinum neurotoxins are suitable for immunoprophylaxis or as immunotherapeutics.

EXAMPLE 4 Dengue Virus

[0182] Human full-length neutralizing antibodies would be particularly useful as logical and natural anti-toxins or anti-infectives as they have already been proven to be safe and well tolerated for other therapeutic purposes. Neutralizing antibodies, either raised by vaccination in animals or passively administered to a variety of animal hosts, have been shown in some instances to provide protection against dengue. However, there are indications that infection with dengue in humans is potentiated by vaccination, and reports that antibodies against specific dengue antigens can themselves cause hemorrhage through cross-reaction with common epitopes on clotting and integrin/adhesin proteins (Falconar, 1997).

[0183] Generation of 16 antibody libraries from blood or bone marrow samples of 8 human donors infected or vaccinated with different serotypes of dengue virus are created. Two libraries are generated from each donor, one utilizing kappa light chains, and the other utilizing lambda light chains. The 8 donors include 4 donors singly infected or vaccinated with each of the four serotypes of dengue, two libraries from individuals infected with multiple dengue serotypes, and two libraries from individuals who have received the tetravalent dengue vaccine. The 16 antibody libraries are used for selection against live cells, live virus, and viral lysates as well as recombinant dengue antigens including envelope and NS1 proteins from the four dengue serotypes.

[0184] The identified Fab antibodies are purified for use in characterization of specificity, affinity, and competition with other Fabs and antibodies.

[0185] Key dengue antibody fragments characterized as neutralizing are converted to whole human IgG1 by subcloning coding regions into in-house mammalian expression vectors. Transfection of plasmids containing whole IgG coding sequences into mammalian cells allows production of large quantities of IgG for use in characterization and passive immunotherapy.

EXAMPLE 5 West Nile Virus

[0186] By applying the previously described library creation and panning technologies, antibodies that bind many of the different West Nile virus strains are isolated and produced in large quantities. As with the neutralizing antibodies described above for anthrax and VEEV, these fully human antibodies against West Nile virus are suitable for immunoprophylaxis or as immunotherapeutics.

EXAMPLE 6 Small Pox/Vaccinia Virus

[0187] By screening the previously described immunized human libraries against an individual antigen known to be involved in the neutralization of vaccinia, a similar panel of antibodies that bind to the antigen is obtained, and antibodies capable of neutralizing viral entry and spread in vitro and in vivo are identified. Furthermore, many variants of similar heavy chain/light chain pairs are identified by the techniques described herein, providing a range of affinities from which to select the candidates with the most desirable characteristics for testing and development. Use of more complicated mixtures of antigens for selection, such as infected cells, lysates, or virions, is also contemplated as an alternative approach. High affinity candidates derived in accordance with this can be used alone for immunoprophylaxis, without the need for affinity maturation that some other approaches may require. Alternatively, a cocktail of antibodies against specific antigens can be used, if desired. For example, Hooper et al. (2000) found that DNA vaccination utilizing genes L1R and A33R of vaccinia was more efficacious than either alone, indicating that for these two antigens, antibodies raised against both gave better protection than antibodies against one. Nowakowski et al., (Nowakowski et al., 2002) found that a mixture of three antibodies to non-overlapping epitopes derived by phage display produced potent neutralization of the botulinum neurotoxin, where each antibody alone showed little effect.

EXAMPLE 7 Elisa Assay to Detect the Presence of Human Antibodies Against Toxins in Patient Fluids

[0188] In this assay, 50 μl of a 4 ng/ml solution of the antigen PA83 (List Laboratories) in Solution A (PBS+0.08% BC solution) are bound to Immulon 2 HB plates (VWR) and allowed to incubate overnight at 4° C. BC solution is PBS containing 0.5% casein (Sigma), 0.01% thimerosal (Sigma), 0.005% phenol red, and 0.01N NaOH. Wells are then washed 3× with PBS+0.05% Tween 20 and blocked by incubation for 1 hour with Solution C (BC solution+1% Tween 20). Wells are washed again 3× with PBS+0.05% Tween 20 and allowed to incubate for one to two hours at 37° C. with 50 μl of patient samples (for example blood, serum, pleural lavage fluids) either straight or as a dilution series. Dilutions are performed in Solution D (BC+0.025% Tween 20). As positive controls, some wells are incubated with the anti-anthrax antibodies described above in Example 1 diluted to concentrations to be determined for this assay in Solution D. Wells are washed 3× with PBS+0.05% Tween 20 and then incubated for one to two hours at 37° C. with a secondary antibody, alkaline-phosphatase labeled goat anti-human F(ab′)₂ in Solution D. Wells are washed 3× with PBS+0.05% Tween 20 and then detected using Phosphatase Substrate (Sigma) in 10 mM diethanolamine, 0.5 mM MgCl₂, pH 9.5. Positive samples are detected and quantified at A 405 using an ELISA reader.

EXAMPLE 8 Assay to Detect and Quantitate PA83 in Patient Fluids

[0189] In this assay, two antibodies against anthrax PA83 are used. The first antibody is bound to a solid substrate (for example, a microtiter plate, beads, or a dipstick). For example, 50 μl of a 4 ng/ml solution of an antibody from Example 1 in Solution A (PBS+0.08% BC solution) is incubated overnight at 4° C. in a microtiter well. Wells are then washed 3× with PBS+0.05% Tween 20 and blocked by incubation for 1 hour with Solution C. Wells are washed again 3× with PBS+0.05% Tween 20 and allowed to incubate for one to two hours at 37° C. with 50 μl of patient samples (for example blood, serum, pleural lavage fluids) either straight or as a dilution series. A standard curve using a dilution series of PA83 is also included. Dilutions are performed in Solution D (BC+0.025% Tween 20). Wells are washed 3× with PBS+0.05% Tween 20 and then incubated for one to two hours at 37° C. with a second anti-anthrax antibody that binds a non-competitive epitope on PA83. This second antibody is labeled with alkaline phosphatase. Wells are washed 3× with PBS+0.05% Tween 20 and then detected using Phosphatase Substrate (Sigma) in 10 mM diethanolamine, 0.5 mM MgCl₂, pH 9.5. Positive samples are detected at A 405 using an ELISA reader.

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[0289] It will be understood that various modifications may be made to the embodiments disclosed herein. For example, as those skilled in the art will appreciate, the specific sequences described herein can be altered slightly without necessarily adversely affecting the functionality of the antibody or antibody fragment. For instance, substitutions of single or multiple amino acids in the antibody sequence can frequently be made without destroying the functionality of the antibody or fragment. Thus, it should be understood that antibodies having a degree of homology greater than 70% to the specific antibodies described herein are within the scope of this disclosure. In particularly useful embodiments, antibodies having a homology greater than about 80% to the specific antibodies described herein are contemplated. In other useful embodiments, antibodies having a homology greater than about 90% to the specific antibodies described herein are contemplated. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of this disclosure.

1 118 1 124 PRT human 1 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Tyr Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Pro Glu 35 40 45 Trp Met Gly Trp Ile Asn Gly Gly Asp Gly Lys Thr Lys Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Leu Ala Ile Thr Arg Asp Thr Ser Ala Arg Thr 65 70 75 80 Ala Tyr Met Glu Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Gly Ala Glu Met Thr Val Gly Ser Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 2 127 PRT human 2 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Ser Trp Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ala Ile Ser Ala Ser Gly Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Gly Thr Leu Val Ala Pro Asp Gly Ser Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 3 134 PRT human 3 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Thr Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 4 134 PRT human 4 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 5 134 PRT human 5 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 6 134 PRT human 6 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Glu Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 7 134 PRT human 7 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 8 134 PRT human 8 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Ala Lys Gly 130 9 134 PRT human 9 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Asn Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 10 134 PRT human 10 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Met Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Pro Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Asn Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 11 137 PRT human 11 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Lys Asp Arg Ile Ile Val Pro Ala Gln Asn His Pro Thr 100 105 110 Gly Phe Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Ala Thr Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 12 135 PRT human 12 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 85 90 95 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Ile Phe Ser 100 105 110 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 115 120 125 Trp Val Ala Val Ile Ser Tyr 130 135 13 135 PRT human 13 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asn Arg Val Ile Val Pro Ala Arg Asn Val Asp Tyr 100 105 110 Ile Tyr Tyr Gly Met Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 14 135 PRT human MISC_FEATURE (26)..(26) xaa=unknown amino acid 14 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Xaa Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Phe Ile Ser Tyr Asp Gly Ser Lys Lys Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asp Arg Val Ile Val Pro Ala Ala Asn Lys Tyr Tyr 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 15 134 PRT human 15 Leu Glu Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Thr Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ser Ile Phe Arg 20 25 30 Thr Ser Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Val Ile Pro Met Val Ser Leu Pro Asp Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr 65 70 75 80 Cys Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Glu Ser Gly Tyr Lys Asp Ser Lys Thr Tyr Tyr 100 105 110 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 16 134 PRT human 16 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Asn Phe Asn 20 25 30 Thr Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Ile Ile Pro Ile Val Gly Ile Ala Asp Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Gly Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Glu Ser Gly Tyr Thr Asn Arg Gly Thr Tyr Tyr 100 105 110 Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 17 134 PRT human 17 Leu Glu Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser 20 25 30 Asn Tyr Ala Thr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Leu Gly Gly Ile Ile Pro Val Phe Gly Thr Ala Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Asn Ser Leu Thr Phe Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Gly Gly Gly Trp Gly Gly Arg Asn Tyr Tyr Tyr 100 105 110 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 18 136 PRT human 18 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg 20 25 30 Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ala Ser Ala Ile Thr Ser Thr Gly Ser Asp Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser 65 70 75 80 Leu Leu Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Pro Gly Arg Gly Tyr Gly Pro Asn Ala Leu Gly 100 105 110 Pro Tyr Phe Tyr Gly Met Asp Val Trp Gly Pro Gly Thr Thr Val Thr 115 120 125 Val Ser Ser Ala Ser Thr Lys Gly 130 135 19 162 PRT human 19 Ser Arg Ala Ile Gln Leu Thr Ser Pro Ser Thr Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Gly 20 25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu 35 40 45 Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr His Cys Gln Gln Tyr Ser Gly Asn Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser 20 163 PRT human 20 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Leu Thr Cys Arg Ala Ser Gln Gly Val Arg 20 25 30 Asn Ala Leu Val Trp Tyr Gln Lys Pro Gly Lys Ala Pro Glu Arg Leu 35 40 45 Ile Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Gly Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser 21 164 PRT human 21 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu 85 90 95 Gly Val Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser 22 166 PRT human 22 Ser Arg Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu 35 40 45 Val Ile Tyr Asp Thr Ser Asn Leu Ala Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ala Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Ser Tyr Asp Lys Phe 85 90 95 Pro Pro Val Phe Asn Phe Gly Pro Gly Thr Thr Val Asp Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser 165 23 164 PRT human 23 Ser Arg Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ile Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Ser Tyr Ser Ala Leu 85 90 95 Gly Phe Thr Phe Gly Pro Gly Thr Thr Val Asp Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser 24 164 PRT human 24 Ser Arg Asp Ile Gln Leu Ala Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Gly Ile Ser 20 25 30 Asn Phe Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Ser Leu Glu Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asp Leu 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Arg Gly Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser 25 165 PRT human 25 Ser Arg Glu Ile Val Met Thr Gln Ser Pro Asp Thr Leu Ser Val Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 20 25 30 Ser Asn Leu Ala Trp Phe Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asn Trp 85 90 95 Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser 165 26 165 PRT human 26 Ser Arg Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Arg Ser Ile Leu 20 25 30 Phe Ser Ser Asn Asn Lys Asn Phe Leu Ala Trp Gln Gln Arg Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Val Ser Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70 75 80 Thr Ile Asp Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln 85 90 95 Gln Tyr Tyr Ser Thr Pro His Thr Phe Gly Gln Gly Thr Lys Leu Glu 100 105 110 Ile Lys Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 115 120 125 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 130 135 140 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 145 150 155 160 Leu Gln Ser Gly Asn 165 27 165 PRT human 27 Ser Arg Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly 20 25 30 Arg Asn Arg Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Leu Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Glu Asp Glu Gly Asp Tyr Tyr Cys Ala Ala Trp Asp Asp 85 90 95 Ser Leu His Gly Gly Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 Gly Ser Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu 165 28 166 PRT human 28 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Asn Asn Ile Glu Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp 85 90 95 Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val Lys Ala Gly Val Glu 165 29 165 PRT human 29 Ser Arg Ser Val Leu Thr Gln Pro Pro Ser Thr Ser Gly Thr Pro Gly 1 5 10 15 Gln Ser Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser 20 25 30 Asn Thr Val Asn Trp Tyr Arg His Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asp Asn Leu Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Glu Thr 85 90 95 Leu Asn Gly Val Ile Tyr Gly Gly Gly Thr Lys Leu Thr Ala Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu 165 30 166 PRT human 30 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Ser Ser Leu Ser Gly Ser Thr Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Thr Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val Lys Ala Gly Val 165 31 166 PRT human 31 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Ser Ser Leu Ser Gly Leu Val Phe Gly Gly Gly Thr Arg Leu Thr Ala 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val Lys Ala Gly Val 165 32 166 PRT human MISC_FEATURE (12)..(12) xaa=unknown amino acid 32 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Xaa Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Asp Ser Asn Ile Gly 20 25 30 Ser Gly Tyr His Val His Trp Tyr Gln His Leu Pro Gly Lys Gly Pro 35 40 45 Lys Leu Leu Ile Ala Asn Asn Asp Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Glu Phe Ser Ala Phe Leu Ala Ile Thr 65 70 75 80 Gly Leu His Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Asn Thr Leu Pro Gly Ser Leu Phe Gly Gly Gly Thr Arg Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val Lys Ala Gly Val 165 33 165 PRT human 33 Ser Arg Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Ser Ile Thr Ile Ser Cys Thr Gly Thr Arg Asn Asp Val Gly Ser 20 25 30 Tyr Asn Leu Val Ser Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Lys 35 40 45 Leu Ile Ile Tyr Ala Asp Asn Gln Arg Pro Ser Gly Glu Tyr Asn Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Arg Ala Glu Asp Glu Ala Asp Tyr Phe Cys Cys Ser Tyr Ser Leu 85 90 95 Thr Asn Asp Val Ile Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu 165 34 165 PRT human 34 Ser Arg Gln Ser Val Leu Thr Pro Ala Ser Val Ser Gly Ser Pro Gly 1 5 10 15 Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser 20 25 30 Tyr Lys Leu Val Ser Trp Tyr Gln Gln His Pro Asp Lys Ala Pro Lys 35 40 45 Leu Ile Ile Tyr Glu Ile Asn Gln Arg Pro Ser Gly Val Ser Asp Arg 50 55 60 Phe Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Thr Asp 85 90 95 Ile Pro Ser Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu 165 35 166 PRT human 35 Ser Arg Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly 20 25 30 Ala Tyr Asn Tyr Val Ser Trp Tyr His His His Pro Gly Lys Ala Pro 35 40 45 Lys Leu Met Ile Tyr Asp Val Tyr Arg Arg Pro Ser Trp Val Ser Ser 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Ile Ser Tyr Thr 85 90 95 Thr Arg Asp Thr Leu Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val Lys Ala Gly Val Glu 165 36 166 PRT human 36 Ser Arg Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Val Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly 20 25 30 Gly Tyr Asn His Val Ser Gln Tyr Gln Gln Tyr Pro Gly Lys Ala Pro 35 40 45 Lys Leu Met Ile Ser Asp Val Asn Lys Arg Pro Ser Gly Val Ser Asn 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr 85 90 95 Asn Thr Asn Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val Lys Ala Gly Val Glu 165 37 166 PRT human 37 Ser Arg Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gln Ser Pro 1 5 10 15 Gly Lys Thr Val Ile Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Val 20 25 30 Gly Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr 35 40 45 Thr Leu Ile Tyr Lys Gly Asn Gln Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile 65 70 75 80 Ser Gly Leu Glu Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr 85 90 95 Asp Ser Ser Tyr Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val Lys Ala Gly Val Glu 165 38 165 PRT human 38 Ser Arg Gln Ala Val Leu Thr Pro Ser Ser Leu Ser Ala Ser Pro Gly 1 5 10 15 Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Phe Tyr Val Gly 20 25 30 Ser Tyr Met Ile Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln 35 40 45 Phe Leu Leu Arg Tyr Arg Ser Asp Ser Asp Ile Gln Arg Gly Ser Gly 50 55 60 Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Thr Ser Ala Asn Ala Gly 65 70 75 80 Ile Leu Leu Ile Ser Gly Leu Gln Pro Glu Asp Glu Ala Asp Tyr Tyr 85 90 95 Cys Met Ile Trp His Ile Asp Thr Val Phe Phe Gly Gly Gly Ser Lys 100 105 110 Leu Thr Val Leu Gly Gln Ser Lys Ala Ala Pro Ser Val Thr Leu Phe 115 120 125 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 130 135 140 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala 145 150 155 160 Asp Ser Ser Pro Val 165 39 114 PRT human 39 Ser Arg Glu Ile Val Met Thr Gln Ser Pro Asp Thr Leu Ser Val Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 20 25 30 Ser Asn Leu Ala Trp Phe Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asn Trp 85 90 95 Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr 100 105 110 Val Ala 40 113 PRT human 40 Ser Arg Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg 20 25 30 Thr Asn Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Leu Ser Ser Leu 65 70 75 80 Gln Ser Glu Asp Phe Ala Leu Tyr Phe Cys Gln His Tyr Asp Ser Trp 85 90 95 Pro Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala 41 109 PRT human MISC_FEATURE (5)..(5) xaa=unknown amino acid 41 Ser Arg Asp Ile Xaa Met Thr Gln Ser Pro Ser Thr Leu Ser Xaa Ser 1 5 10 15 Xaa Gly Glu Arg Ala Thr Leu Ser Cys Xaa Ala Ser Gln Ser Val Ser 20 25 30 Xaa Xaa Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ser Arg Phe 50 55 60 Xaa Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Ser Glu Asp Phe Ala Xaa Tyr Tyr Cys Gln Gln Tyr Lys Lys Xaa 85 90 95 Xaa Pro Xaa Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 42 112 PRT human 42 Ser Arg Ala Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 20 25 30 Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr His Cys Gln Gln Tyr Ser Gly Asn 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 43 106 PRT human 43 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 1 5 10 15 Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Gly Trp Leu Ala Trp Tyr 20 25 30 Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr Lys Ala Ser 35 40 45 Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 50 55 60 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 65 70 75 80 Thr Tyr His Cys Gln Gln Tyr Ser Gly Asn Trp Thr Phe Gly Gln Gly 85 90 95 Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 44 113 PRT human 44 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr 20 25 30 Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Ala Ser Ile Leu Glu Ser Gly Val Ser Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Asp Tyr 85 90 95 Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala 45 113 PRT human 45 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr 20 25 30 Arg Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Ala Ser Ile Leu Glu Ser Gly Val Ser Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Arg Gln Phe Asn Asp Tyr 85 90 95 Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala 46 113 PRT human 46 Ser Arg Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr 20 25 30 Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Ala Ser Ile Leu Glu Ser Gly Val Ser Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Asp Tyr 85 90 95 Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala 47 113 PRT human 47 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Leu Thr Cys Arg Ala Ser Gln Gly Val Arg 20 25 30 Asn Ala Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Arg 35 40 45 Leu Ile Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Gly Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr 85 90 95 Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala 48 113 PRT human 48 Ser Arg Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser 20 25 30 Thr Trp Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Thr Asn Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ala Val 85 90 95 Pro Pro Ala Phe Gly Gly Gly Thr Lys Val Glu Met Asn Arg Thr Val 100 105 110 Ala 49 113 PRT human 49 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu 85 90 95 Gly Val Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105 110 Ala 50 115 PRT human 50 Ser Arg Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu 35 40 45 Val Ile Tyr Asp Thr Ser Asn Leu Ala Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ala Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Ser Tyr Asp Lys Phe 85 90 95 Pro Pro Val Phe Asn Phe Gly Pro Gly Thr Thr Val Asp Ile Lys Arg 100 105 110 Thr Val Ala 115 51 112 PRT human 51 Ser Arg Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ala 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Val Ser Asn Leu Glu Pro Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Asn Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr His Asn Leu 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 52 113 PRT human 52 Ser Arg Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn 20 25 30 Asn His Leu Asn Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Val 35 40 45 Leu Ile Tyr Asp Val Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Phe Cys His Gln Tyr Glu Ser Val 85 90 95 Arg Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala 53 113 PRT human 53 Ser Arg Asp Ile Gln Leu Ala Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Gly Ile Ser 20 25 30 Asn Phe Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Ser Leu Glu Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asp Leu 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Arg Gly Thr Val 100 105 110 Ala 54 114 PRT human 54 Ser Arg Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Ser Tyr Ser Ala 85 90 95 Leu Gly Phe Thr Phe Gly Pro Gly Thr Thr Val Asp Ile Lys Arg Thr 100 105 110 Val Ala 55 113 PRT human 55 Ser Arg Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ser Leu 35 40 45 Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Arg Thr 85 90 95 Ser Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala 56 113 PRT human 56 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser 20 25 30 Gly Ser Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Tyr Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Gly Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Ser Asn 85 90 95 Thr Val Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105 110 Ala 57 113 PRT human 57 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Asn Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Leu Arg Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Ser Asn Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Ile 85 90 95 Pro Met Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg Thr Val 100 105 110 Ala 58 113 PRT human 58 Ser Arg Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Tyr Arg Pro Gly Arg Ala Pro Asn Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Tyr Arg Gly Ala Pro Ala Arg Phe 50 55 60 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Thr Asn Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Thr 85 90 95 Pro Met Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Glu Arg Thr Val 100 105 110 Ala 59 113 PRT human 59 Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr 20 25 30 Lys Asn Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Asn Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Leu Arg Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Thr 85 90 95 Pro Met Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg Thr Val 100 105 110 Ala 60 113 PRT human 60 Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala 1 5 10 15 Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr 20 25 30 Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu 35 40 45 Leu Ile Tyr Met Gly Ser Ser Arg Ala Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val 65 70 75 80 Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Pro Leu Gln Thr 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala 61 104 PRT human 61 Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala 1 5 10 15 Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr 20 25 30 Asn Tyr Val Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu 35 40 45 Leu Ile Tyr Met Gly Ser Ser Arg Ala Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile Ser Arg Val 65 70 75 80 Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Pro Leu Gln Thr 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr 100 62 162 PRT human 62 Ser Arg Gln Ser Ala Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly 20 25 30 Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro 35 40 45 Lys Leu Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Pro Asn 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr 85 90 95 Ser Ser Ser Thr Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val 63 161 PRT human 63 Ser Arg Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly 20 25 30 Ala Tyr Asn Tyr Val Ser Trp Tyr His His His Pro Gly Lys Ala Pro 35 40 45 Lys Leu Met Ile Tyr Asp Val Tyr Arg Arg Pro Ser Trp Val Ser Ser 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Ile Ser Tyr Thr 85 90 95 Thr Arg Asp Thr Leu Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 64 161 PRT human 64 Ser Arg Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly 20 25 30 Ser Tyr Lys Leu Val Ser Trp Tyr Gln Gln His Pro Asp Lys Ala Pro 35 40 45 Lys Leu Ile Ile Tyr Glu Ile Asn Gln Arg Pro Ser Gly Val Ser Asp 50 55 60 Arg Phe Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Thr 85 90 95 Asp Ile Pro Ser Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 65 161 PRT human 65 Ser Arg Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Arg Asn Asp Val Gly 20 25 30 Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro 35 40 45 Lys Leu Ile Ile Tyr Ala Asp Asn Gln Arg Pro Ser Gly Glu Tyr Asn 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Arg Ala Glu Asp Glu Ala Asp Tyr Phe Cys Cys Ser Tyr Ser 85 90 95 Leu Thr Asn Asp Val Ile Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 66 162 PRT human 66 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Ser Ser Leu Ser Gly Leu Val Phe Gly Gly Gly Thr Arg Leu Thr Ala 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val 67 162 PRT human 67 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Ser Ser Leu Ser Gly Ser Thr Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Thr Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val 68 162 PRT human MISC_FEATURE (12)..(12) xaa=unknown amino acid 68 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Xaa Ser Gly Ala Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Asp Ser Asn Ile Gly 20 25 30 Ser Gly Tyr His Val His Trp Tyr Gln His Leu Pro Gly Lys Gly Pro 35 40 45 Lys Leu Leu Ile Ala Asn Asn Asp Asn Arg Pro Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Lys Ser Glu Phe Ser Ala Phe Leu Ala Ile Thr 65 70 75 80 Gly Leu His Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp 85 90 95 Asn Thr Leu Pro Gly Ser Leu Phe Gly Gly Gly Thr Arg Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val 69 161 PRT human 69 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Asn Asn Ile Glu Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp 85 90 95 Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 70 116 PRT human 70 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Asn Asn Ile Glu Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp 85 90 95 Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys 115 71 151 PRT human 71 Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly 1 5 10 15 Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu 20 25 30 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asn Asn Ile Glu Arg Pro 35 40 45 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala 50 55 60 Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr 65 70 75 80 Cys Ala Thr Trp Asp Asp Ser Leu Asn Gly Val Val Phe Gly Gly Gly 85 90 95 Thr Gln Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr 100 105 110 Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu 115 120 125 Val Cys Leu Val Ser Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp 130 135 140 Lys Ala Asp Ser Ser Pro Val 145 150 72 161 PRT human 72 Ser Arg Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly 20 25 30 Arg Asn Arg Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Leu Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Glu Asp Glu Gly Asp Tyr Tyr Cys Ala Ala Trp Asp Asp 85 90 95 Ser Leu His Gly Gly Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 Gly Gln Ser Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 73 162 PRT human 73 Ser Arg Gln Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro 1 5 10 15 Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30 Thr Asn Ser Val Thr Trp Tyr Gln Arg Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Gly Ser His Gln Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly 65 70 75 80 Leu Gln Ser Gly Asp Glu Ala Asp Tyr Phe Cys Val Val Trp Asp Asp 85 90 95 Asn Leu Asn Gly Ala Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser 115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser 145 150 155 160 Pro Val 74 161 PRT human 74 Ser Arg Gln Ser Val Leu Thr Gln Pro Pro Ser Thr Ser Gly Thr Pro 1 5 10 15 Gly Gln Ser Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30 Ser Asn Thr Val Asn Trp Tyr Arg His Leu Pro Gly Thr Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Gly Asp Asn Leu Arg Pro Ser Gly Val Ser Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly 65 70 75 80 Leu Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Glu 85 90 95 Thr Leu Asn Gly Val Ile Tyr Gly Gly Gly Thr Lys Leu Thr Ala Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 75 157 PRT human 75 Ser Arg Ser Tyr Val Leu Thr Gln Pro Pro Ser Glu Ser Val Ala Pro 1 5 10 15 Gly Gln Thr Ala Arg Ile Ser Cys Gly Gly Ser Asn Ile Gly Ser Tyr 20 25 30 Gly Val His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val 35 40 45 Val His Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser 50 55 60 Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Ser Ala 85 90 95 Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 76 161 PRT human 76 Ser Arg Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gln Ser Pro 1 5 10 15 Gly Lys Thr Val Ile Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Val 20 25 30 Gly Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr 35 40 45 Thr Leu Ile Tyr Lys Gly Asn Gln Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile 65 70 75 80 Ser Gly Leu Glu Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr 85 90 95 Asp Ser Ser Tyr Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 145 150 155 160 Val 77 166 PRT human 77 Ser Arg Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro 1 5 10 15 Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Phe Tyr Val 20 25 30 Gly Ser Tyr Met Ile Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro 35 40 45 Gln Phe Leu Leu Arg Tyr Arg Ser Asp Ser Asp Ile Gln Arg Gly Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Thr Ser Ala Asn Ala 65 70 75 80 Gly Ile Leu Leu Ile Ser Gly Leu Gln Pro Glu Asp Glu Ala Asp Tyr 85 90 95 Tyr Cys Met Ile Trp His Ile Asp Thr Val Phe Phe Gly Gly Gly Ser 100 105 110 Lys Leu Thr Val Leu Gly Gln Ser Lys Ala Ala Pro Ser Val Thr Leu 115 120 125 Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val 130 135 140 Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Leu Thr Val Ala Trp Lys 145 150 155 160 Ala Asp Ser Ser Pro Val 165 78 133 PRT human 78 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Phe Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Asn Ile Lys Gln Asp Gly Ser Gly Lys Tyr Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ala Arg Thr Asn Gly Gly Tyr Asp Ile Tyr Tyr Tyr 100 105 110 Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 Ala Ser Thr Lys Gly 130 79 121 PRT human 79 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Pro Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Thr Asp Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Cys Val Ala Asn Ile Asn Gln Asp Gly Ser Glu Arg Tyr Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg Asn Ala Arg Gly Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 80 136 PRT human 80 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg 20 25 30 Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ala Ser Ala Ile Thr Ser Thr Gly Ser Asp Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser 65 70 75 80 Leu Leu Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Pro Gly Arg Gly Tyr Gly Pro Asn Ala Leu Gly 100 105 110 Pro Tyr Phe Tyr Gly Met Asp Val Trp Gly Pro Gly Thr Thr Val Thr 115 120 125 Val Ser Ser Ala Ser Thr Lys Gly 130 135 81 132 PRT human 81 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Asp Tyr Ser Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Ser Tyr Ile Thr Gly Ser Gly Ser Ala Ile Ser Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser 65 70 75 80 Leu Phe Gln Gln Met Asn Ser Leu Gly Ala Glu Gly Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Glu Tyr Gly Asp Ser Leu Leu Tyr Phe Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly 130 82 126 PRT human 82 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ser Ile Thr Asp Arg Gly Thr Tyr Lys Asp Tyr Ala Glu 50 55 60 Ser Leu Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser 65 70 75 80 Met Ser Leu Gln Met Asp Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr 85 90 95 Phe Cys Ala Arg Glu Arg Phe Gly Asp Asp Gly Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 83 130 PRT human 83 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Glu Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Pro 20 25 30 Asp Tyr Ala Met His Trp Val Arg Gln Arg Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Val Ile Asn Trp Asn Gly Ile Thr Arg Arg Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Ile Asp Arg Asp Ser Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Asp Gly Gly Gly Asn Gly Gly Ser Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Ile Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly 130 84 134 PRT human 84 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Glu Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 85 134 PRT human 85 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 86 134 PRT human 86 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 87 134 PRT human 87 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Thr Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 88 134 PRT human 88 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 89 134 PRT human 89 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Ala Lys Gly 130 90 134 PRT human 90 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Ala Ile Ser Tyr Asp Gly Thr Asn Lys Asp Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Val 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Lys Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 91 134 PRT human 91 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Met Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Pro Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Asn Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 92 134 PRT human MISC_FEATURE (40)..(40) xaa=unknown amino acid 92 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Thr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Xaa Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Pro Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Xaa Ala Lys Glu Gly Val Ile Ile Pro Xaa Ser Thr Asn Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 93 134 PRT human 93 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Asn Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 94 134 PRT human MISC_FEATURE (81)..(81) xaa=unknown amino acid 94 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Xaa Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Glu Gly Val Ile Ile Pro Ala Ala Thr Xaa Asp Arg 100 105 110 Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 95 135 PRT human 95 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asn Arg Val Ile Val Pro Ala Arg Asn Val Asp Tyr 100 105 110 Ile Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 96 135 PRT human 96 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asn Arg Val Ile Val Pro Ala Arg Asn Val Asp Tyr 100 105 110 Ile Tyr Tyr Gly Met Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 97 135 PRT human 97 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Leu Ile Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Thr Asp Thr Ala Val Tyr 85 90 95 Phe Cys Ala Lys Asp Arg Val Ile Val Pro Ala Ala Asn Asp Tyr Tyr 100 105 110 Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 98 135 PRT human MISC_FEATURE (26)..(26) xaa=unknown amino acid 98 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Xaa Ser Gly Phe Ile Phe Ser 20 25 30 Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Phe Ile Ser Tyr Asp Gly Ser Lys Lys Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asp Arg Val Ile Val Pro Ala Ala Asn Lys Tyr Tyr 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly 130 135 99 137 PRT human 99 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg 20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Gly Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Lys Asp Arg Ile Ile Val Pro Ala Gln Asn His Pro Thr 100 105 110 Gly Phe Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Ala Thr Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 100 134 PRT human MISC_FEATURE (101)..(101) xaa=unknown amino acid 100 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Gly 20 25 30 Ser Tyr Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Asn Lys Thr 65 70 75 80 Leu Phe Leu Gln Met Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Xaa Gly Val Ile Val Pro Ala Ser Thr Arg Thr Arg 100 105 110 Tyr Tyr Arg Met Asp Val Trp Gly Gln Gly Thr Ala Val Ser Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 101 129 PRT human 101 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Ala Phe Thr Phe Arg 20 25 30 Asp Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Leu Ile Ser His Asp Gly Ala Gln Thr Ser Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Glu Thr 65 70 75 80 Val Phe Leu Gln Met Ser Gly Leu Arg Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ser Lys Ser Thr Ala Lys Thr Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Pro Gly Thr Ala Val Thr Ala Ser Ser Ala Ser Thr Lys 115 120 125 Gly 102 138 PRT human 102 Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asp 20 25 30 Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Arg Glu 35 40 45 Trp Val Ser Leu Ile Ser Glu Asp Gly Tyr Asn Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Gly Pro Glu Asp Thr Ala Phe Tyr 85 90 95 Phe Cys Ala Lys Ala Gly Pro Gln Arg Arg Tyr Tyr Asp Arg Leu Pro 100 105 110 Gly Tyr Tyr Pro Ser Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu 115 120 125 Val Ser Val Ser Ser Ala Ser Thr Lys Gly 130 135 103 127 PRT human 103 Leu Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Ser Trp Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ala Ile Ser Ala Ser Gly Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Gly Thr Leu Val Ala Pro Asp Gly Ser Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 104 133 PRT human 104 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Thr Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Gly Ile Ser Ala Arg Ser Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Met Tyr Val Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Arg Tyr Lys Leu Ala Tyr Cys Thr Gly Asp Cys Tyr Pro 100 105 110 Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 Ala Ser Thr Lys Gly 130 105 132 PRT human 105 Leu Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Pro Cys Val Val Ser Gly Tyr Thr Leu Arg 20 25 30 Asn Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Val Ser Gly Ser Gly Ser Asp Ala Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ser Lys Asp Thr 65 70 75 80 Leu Phe Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Tyr Ser Ser Gly Trp Asp Phe Tyr His Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly 130 106 126 PRT human 106 Leu Glu Gln Met Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser 20 25 30 Asp His Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp 35 40 45 Trp Val Gly Arg Thr Lys Asn Lys Ala Asn Ser Tyr Thr Ile Glu Tyr 50 55 60 Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 65 70 75 80 Asn Ser Phe Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Trp Thr Ser Gly Ala Cys Asn Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 107 124 PRT human 107 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Tyr Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Pro Glu 35 40 45 Trp Met Gly Trp Ile Asn Gly Gly Asp Gly Lys Thr Lys Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Leu Ala Ile Thr Arg Asp Thr Ser Ala Arg Thr 65 70 75 80 Ala Tyr Met Glu Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Gly Ala Glu Met Thr Val Gly Ser Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 108 136 PRT human 108 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Trp Ile Asn Pro Asn Thr Gly Asp Thr Asn Tyr Ala Gln 50 55 60 Asn Phe Gln Gly Trp Val Thr Met Thr Arg Asp Thr Ser Ile Arg Thr 65 70 75 80 Ala Tyr Thr Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Phe Tyr Ser Asp Ser Ser Gly Tyr Tyr Tyr Gly 100 105 110 Tyr Tyr Ser Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser Ala Ser Thr Lys Gly 130 135 109 134 PRT human 109 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Asn Phe Asn 20 25 30 Thr Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Ile Ile Pro Ile Val Gly Ile Ala Asp Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Glu Ser Gly Tyr Thr Asn Arg Gly Thr Tyr Tyr 100 105 110 Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 110 134 PRT human 110 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Asn Phe Asn 20 25 30 Thr Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Ile Ile Pro Ile Val Gly Ile Ala Asp Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Gly Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Glu Ser Gly Tyr Thr Asn Arg Gly Thr Tyr Tyr 100 105 110 Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 111 134 PRT human 111 Leu Glu Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser 20 25 30 Asn Tyr Ala Thr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Leu Gly Gly Ile Ile Pro Val Phe Gly Thr Ala Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Asn Ser Leu Thr Phe Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Gly Gly Gly Trp Gly Gly Arg Asn Tyr Tyr Tyr 100 105 110 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly 130 112 128 PRT human 112 Leu Glu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Glu Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 Thr His Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu 35 40 45 Trp Met Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Lys Tyr Gly Pro 50 55 60 Ser Phe Gln Gly Glu Val Thr Ile Ser Cys Asp Lys Ser Ile Asn Thr 65 70 75 80 Ala Tyr Leu Gln Trp Arg Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Arg Tyr Arg Arg Gln Leu Asn Pro Tyr Ala Phe Asp Met 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Leu Ala Ser Thr Lys Gly 115 120 125 113 110 PRT human 113 Ser Arg Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 1 5 10 15 Pro Gly Asp Thr Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly 20 25 30 Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile His Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Gly Arg Phe 50 55 60 Ser Gly Ala Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Ser Asp Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Asn Trp 85 90 95 Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 114 112 PRT human 114 Ser Arg Gln Ser Ala Leu Thr Gln Pro His Ser Ala Ser Gly Pro Pro 1 5 10 15 Asp Gln Thr Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Glu 20 25 30 Gly Asn Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Gln 35 40 45 Leu Leu Ile Tyr Gly Lys Asp Gln Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp 85 90 95 Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 115 110 PRT human 115 Ser Arg Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro 1 5 10 15 Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys 20 25 30 Ser Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val 35 40 45 Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr His Cys Gln Val Trp Asp Ser Ser Ser 85 90 95 Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 116 123 PRT human 116 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Val Gly Thr Asn 20 25 30 Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Ile Gly Gly Thr Thr Tyr Glu Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ser Gln Leu Trp Phe Gly Glu Leu Phe Gly His Asp Val Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Val Val Thr Val Ser Ser 115 120 117 129 PRT human MISC_FEATURE (66)..(66) xaa=unknown amino acid 117 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Arg Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ala Val Ile Ser His Asp Gly Ser His Glu Glu Tyr Ala Asp Ser Gly 50 55 60 Lys Xaa Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Xaa Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Tyr Tyr Tyr Asp Tyr Ser Gly Tyr Pro Tyr Asp 100 105 110 Tyr Asn Gly Ile Asp Val Trp Gly Gln Gly Thr Thr Val Val Val Ser 115 120 125 Ser 118 123 PRT human 118 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Val Gly Thr Asn 20 25 30 Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Ile Gly Gly Thr Thr Tyr Glu Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ser Gln Leu Trp Phe Gly Glu Leu Phe Gly His Asp Val Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Val Val Thr Val Ser Ser 115 120 

We claim:
 1. A method for treating an animal infected with Bacillus anthracis comprising administering an antibody or antibody fragment having binding affinity of at least 1×10⁻⁸ M to the protective antigen of Bacillus anthracis and the ability to block binding of the protective antigen to one or more members of the group consisting of cell receptors, edema factor and lethal factor.
 2. A method for treating an animal infected with Bacillus anthracis comprising administering an antibody or antibody fragment having a binding affinity of at least 1×10⁻⁸M to a molecule involved in anthrax infection and the ability to block binding of said molecule involved in anthrax infection to one or more members of the group consisting of cell receptors, PA63, PA63 heptamer, PA83, edema factor and lethal factor.
 3. A method for treating an animal infected with Bacillus anthracis comprising administering an antibody or antibody fragment having the ability to prevent EF and/or LF from binding to the PA63 heptamer.
 4. The method of claim 1 wherein the antibody or antibody fragment prevents PA63 from forming a heptamer.
 5. The method of claim 1 wherein the antibody or antibody fragment prevents PA63 from binding to EF or LF.
 6. The method of claim 1 wherein the antibody or antibody fragment prevents EF and/or LF from binding to the PA63 heptamer.
 7. The method of claim 1 wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 1 to
 18. 8. The method of claim 1 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 19 to
 26. 9. The method of claim 1 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 27 to
 38. 10. The method of claim 1 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 39 to
 61. 11. The method of claim 1 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 62 to
 77. 12. The method of claim I wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 78 to
 112. 13. A method for determining exposure to Bacillus anthracis comprising: obtaining a test sample of a body fluid or tissue from a subject; and assaying for the presence of one or more molecules of the group consisting of cell receptors, PA63, PA63 heptamer, PA83, edema factor and lethal factor in the test sample with an antibody or antibody fragment having binding affinity for said molecule, wherein the presence of elevated levels of said antibody or antibody fragment in the sample correlates with the presence of a disease associated with Bacillus anthracis.
 14. The method of claim 13 wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 1 to
 18. 15. The method of claim 13 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 19 to
 26. 16. The method of claim 13 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 27 to
 38. 17. The method of claim 13 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 39 to
 61. 18. The method of claim 13 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 62 to
 77. 19. The method of claim 13 wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 78 to
 112. 20. A method for determining exposure to Venezuelan equine encephalitis comprising: obtaining a test sample of a body fluid or tissue from a subject; and assaying for the presence of one or more molecules involved in infection by Venezuelan equine encephalitis in the test sample with an antibody or antibody fragment having binding affinity for said molecule, wherein the presence of elevated levels of said antibody or antibody fragment in the sample correlates with the presence of a disease associated with Venezuelan equine encephalitis.
 21. The method of claim 20 wherein the antibody or antibody fragment comprises a variable light chain region selected from the group consisting of SEQ ID NO. 113 to
 115. 22. The method of claim 20 wherein the antibody or antibody fragment comprises a variable heavy chain region selected from the group consisting of SEQ ID NO. 116 to
 118. 23. A method for determining exposure to Bacillus anthracis comprising: obtaining a test sample of a body fluid or tissue from a subject; and assaying for the presence of an antibody to one or more molecules of the group consisting of cell receptors, PA63, PA63 heptamer, PA83, edema factor and lethal factor in the test sample with a secondary antibody or antibody fragment having binding affinity for said antibody, wherein the presence of elevated levels of said secondary antibody or antibody fragment in the sample correlates with the presence of Bacillus anthracis in the subject.
 24. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO.1 to
 18. 25. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO.19 to
 26. 26. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 27 to
 38. 27. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 39 to
 61. 28. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 62 to
 77. 29. The method of claim 23 further comprising correlating the levels of antibody or antibody fragment in the test sample with a control antibody or antibody fragment comprising a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 78 to
 112. 30. A method for determining exposure to Venezuelan equine encephalitis comprising: obtaining a test sample of a body fluid or tissue from a subject; and assaying for the presence of an anti-Venezuelan equine encephalomyelitis virus antibody in the test sample with a secondary antibody or antibody fragment having binding affinity for said antibody, wherein the presence of elevated levels of said secondary antibody or antibody fragment in the sample correlates with the presence of Venezuelan equine encephalitis in the subject.
 31. The method of claim 30 wherein the antibody or antibody fragment comprises a variable light chain region selected from the group consisting of SEQ ID NO. 113 to
 115. 32. The method of claim 30 wherein the antibody or antibody fragment comprises a variable heavy chain region selected from the group consisting of SEQ ID NO. 116to118.
 33. A diagnostic kit for determining exposure to Bacillus anthracis, said kit comprising an antibody that specifically reacts with of one or more molecules involved in anthrax infection of the group consisting of cell receptors, PA63, PA63 heptamer, PA83, edema factor and lethal factor.
 34. The diagnostic kit of claim 33 wherein said antibody is a monoclonal antibody.
 35. The method of claim 33 wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 1 to
 18. 36. The method of claim 33 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 19 to
 26. 37. The method of claim 33 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 27 to
 38. 38. The method of claim 33 wherein the antibody or antibody fragment comprises a light chain kappa region having a sequence selected from the group consisting of SEQ ID NO. 39 to
 61. 39. The method of claim 33 wherein the antibody or antibody fragment comprises a light chain lambda region having a sequence selected from the group consisting of SEQ ID NO. 62 to
 77. 40. The method of claim 33 wherein the antibody or antibody fragment comprises a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO. 78 to
 112. 41. A diagnostic kit for detecting exposure to Venezuelan equine encephalitis comprising an antibody that specifically reacts with one or more molecules involved in infection by Venezuelan equine encephalitis.
 42. A diagnostic kit as in claim 41 wherein the antibody comprises a variable light chain region selected from the group consisting of SEQ ID NO. 113 to
 115. 43. A kit as in claim 41 wherein the antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NO. 116 to
 118. 44. A method of prophylactic treatment comprising administering to a subject a composition comprising a multimer of PA63 in a pharmaceutically acceptable carrier.
 45. A method as in claim 44 wherein the multimer of PA63 comprises up to 7 PA63 units.
 46. A method as in claim 44 wherein the multimer of PA63 comprises a heptamer of PA63.
 47. A vaccine comprising a multimer of PA63 in a pharmaceutically acceptable carrier.
 48. A vaccine as in claim 47 wherein the multimer of PA63 comprises up to 7 PA63 units.
 49. A vaccine as in claim 47 wherein the multimer of PA63 comprises a heptamer of PA63. 